Fabs-in-tandem immunoglobulin and uses thereof

ABSTRACT

The present invention provides multivalent and multispecific binding proteins that are capable of binding two or more antigens, or two or more epitopes. The present invention also provides methods of making and using such multivalent and multispecific binding proteins, including methods of using such binding proteins for prevention or treatment of various diseases, or for detecting specific antigens in vitro or in vivo.

INCORPORATION BY REFERENCE

This application claims priority to International Application No.PCT/CN2013/090923, filed Dec. 30, 2013, which is herein incorporated byreference in its entirety.

FIELD OF INVENTION

The present invention relates to multivalent and multispecific bindingproteins, and to methods of making and using multivalent andmultispecific binding proteins.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:EPBI_001_01WO_SeqList_ST25.txt, date recorded: Dec. 2, 2014, file size98 KB).

BACKGROUND OF THE INVENTION

Bispecific or multispecific antibodies have been generated in attemptsto prepare molecules useful for the treatment of various inflammatorydiseases, cancers, and other disorders.

Bispecific antibodies have been produced using the quadroma technology(see Milstein, C. and A. C. Cuello, Nature, 1983. 305(5934): p. 537-40)based on the somatic fusion of two different hybridoma cell linesexpressing murine monoclonal antibodies with the desired specificitiesof the bispecific antibody. Bispecific antibodies can also be producedby chemical conjugation of two different mAbs (see Staerz, U. D., etal., Nature, 1985. 314(6012): p. 628-31). Other approaches have usedchemical conjugation of two different monoclonal antibodies or smallerantibody fragments (see Brennan, M., et al., Science, 1985. 229(4708):p. 81-3).

Another method is the coupling of two parental antibodies with ahetero-bifunctional crosslinker. In particular, two different Fabfragments have been chemically crosslinked at their hinge cysteineresidues in a site-directed manner (see Glennie, M. J., et al., JImmunol, 1987. 139(7): p. 2367-75).

Other recombinant bispecific antibody formats have been developed in therecent past (see Kriangkum, J., et al., Biomol Eng, 2001. 18(2): p.31-40). Amongst them tandem single-chain Fv molecules and diabodies, andvarious derivatives thereof, have been used for the construction ofrecombinant bispecific antibodies. Normally, construction of thesemolecules starts from two single-chain Fv (scFv) fragments thatrecognize different antigens (see Economides, A. N., et al., Nat Med,2003. 9(1): p. 47-52). Tandem scFv molecules (taFv) represent astraightforward format simply connecting the two scFv molecules with anadditional peptide linker. The two scFv fragments present in thesetandem scFv molecules form separate folding entities. Various linkerscan be used to connect the two scFv fragments and linkers with a lengthof up to 63 residues (see Nakanishi, K., et al Annu Rev Immunol, 2001.19: p. 423-74).

In a recent study, in vivo expression by transgenic rabbits and cattleof a tandem scFv directed against CD28 and a melanoma-associatedproteoglycan was reported (see Gracie, J. A., et al., J Clin Invest,1999. 104(10): p. 1393-401). In this construct the two scFv moleculeswere connected by a CH1 linker and serum concentrations of up to 100mg/L of the bispecific antibody were found. A few studies have nowreported expression of soluble tandem scFv molecules in bacteria (seeLeung, B. P., et al., J Immunol, 2000. 164(12): p. 6495-502; Ito, A., etal., J Immunol, 2003. 170(9): p. 4802-9; Karni, A., et al., JNeuroimmunol, 2002. 125(1-2): p. 134-40) using either a very short Ala3linker or long glycine/serine-rich linkers.

In a recent study, phage display of a tandem scFv repertoire containingrandomized middle linkers with a length of 3 or 6 residues enrichedthose molecules which are produced in soluble and active form inbacteria. This approach resulted in the isolation of a preferred tandemscFv molecule with a 6 amino acid residue linker (see Arndt, M. and J.Krauss, Methods Mol Biol, 2003. 207: p. 305-21).

Bispecific diabodies (Db) utilize the diabody format for expression.Diabodies are produced from scFv fragments by reducing the length of thelinker connecting the VH and VL domain to approximately 5 residues (seePeipp, M. and T. Valerius, Biochem Soc Trans, 2002. 30(4): p. 507-11).This reduction of linker size facilitates dimerization of twopolypeptide chains by crossover pairing of the VH and VL domains.Bispecific diabodies are produced by expressing two polypeptide chainswith either the structure VHA-VLB and VHB-VLA (VH-VL configuration) orVLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A recentcomparative study demonstrates that the orientation of the variabledomains can influence expression and formation of active binding sites(see Mack, M., G. Riethmuller, and P. Kufer, Proc Natl Acad Sci USA,1995. 92(15): p. 7021-5).

One approach to force the generation of bispecific diabodies is theproduction of knob-into-hole diabodies (see Holliger, P., T. Prospero,and G. Winter, Proc Natl Acad Sci USA, 1993. 90(14): p. 6444-8.18). Thiswas demonstrated for a bispecific diabody directed against HER2 and CD3.A large knob was introduced in the VH domain by exchanging Val37 withPhe and Leu45 with Trp and a complementary hole was produced in the VLdomain by mutating Phe98 to Met and Tyr87 to Ala, either in theanti-HER2 or the anti-CD3 variable domains. By using this approach theproduction of bispecific diabodies could be increased from 72% by theparental diabody to over 90% by the knob-into-hole diabody.

Single-chain diabodies (scDb) represent an alternative strategy toimprove the formation of bispecific diabody-like molecules (seeHolliger, P. and G. Winter, Cancer Immunol Immunother, 1997. 45(3-4): p.128-30; Wu, A. M., et al., Immunotechnology, 1996. 2(1): p. 21-36).Bispecific single-chain diabodies are produced by connecting the twodiabody-forming polypeptide chains with an additional middle linker witha length of approximately 15 amino acid residues. Consequently, allmolecules with a molecular weight corresponding to monomericsingle-chain diabodies (50-60 kDa) are bispecific. Several studies havedemonstrated that bispecific single chain diabodies are expressed inbacteria in soluble and active form with the majority of purifiedmolecules present as monomers (see Holliger, P. and G. Winter, CancerImmunol Immunother, 1997. 45(3-4): p. 128-30; Wu, A. M., et al.,Immunotechnology, 1996. 2(1): p. 21-36; Pluckthun, A. and P. Pack,Immunotechnology, 1997. 3(2): p. 83-105; Ridgway, J. B., et al., ProteinEng, 1996. 9(7): p. 617-21).

Diabody have been fused to Fc to generate more Ig-like molecules, nameddi-diabody (see Lu, D., et al., J Biol Chem, 2004. 279(4): p. 2856-65).In addition, multivalent antibody construct comprising two Fab repeatsin the heavy chain of an IgG and capable of binding four antigenmolecules has been described (see U.S. Pat. No. 8,722,859 B2, andMiller, K., et al., J Immunol, 2003. 170(9): p. 4854-61).

The most recent examples are tetravalent IgG-single-chain variablefragment (scFv) fusions (Dong J, et al. 2011 MAbs 3:273-288; Coloma M J,Morrison S L 1997 Nat Biotechnol 15:159-163; Lu D, et al. 2002 J ImmunolMethods 267:213-226), catumaxomab, a trifunctional rat/mouse hybridbispecific epithelial cell adhesion molecule-CD3 antibody (Lindhofer H,et al 1995 J Immunol 155:219-225), the bispecific CD19-CD3 scFv antibodyblinatumomab (Bargou R, et al. 2008 Science 321:974-977), “dual-actingFab” (DAF) antibodies (Bostrom J, et al. 2009 Science 323:1610-1614),covalently linked pharmacophore peptides to catalytic anti-bodies(Doppalapudi V R, et al. 2010 Proc Natl Acad Sci USA 107:22611-22616),use of the dynamic exchange between half IgG4 molecules to generatebispecific antibodies (van der Neut Kolfschoten M, et al. 2007 Science317:1554-1557; Stubenrauch K, et al. 2010 Drug Metab Dispos 38:84-91),or by exchange of heavy-chain and light-chain domains within the antigenbinding fragment (Fab) of one half of the bispecific antibody (CrossMabformat) (Schaefer W et al 2011 Proc Natl Acad Sci 108:11187-92).

There is a need in the art for single molecular entities with dualantigen binding function, and for methods of generating such multivalentand multispecific binding proteins. The present invention addressesthese and other needs.

SUMMARY OF THE INVENTION

The present invention provides multivalent and multispecific bindingproteins, and methods of making and using such binding proteins. In oneembodiment, the multivalent and multispecific binding proteins providedherein are Fabs-in-tandem immunoglobulins (FIT-Ig), and are capable ofbinding two or more antigens, or two or more epitopes of the sameantigen, or two or more copies of the same epitope. The multivalent andmultispecific binding proteins provided herein are useful for treatmentand/or prevention of acute and chronic inflammatory diseases anddisorders, autoimmune diseases, cancers, spinal cord injuries, sepsis,and other diseases, disorders, and conditions. Pharmaceuticalcompositions comprising the multivalent and multispecific bindingproteins are provided herein. In addition, nucleic acids, recombinantexpression vectors, and host cells for making such FIT-Igs are providedherein. Methods of using the FIT-Igs of the invention to detect specificantigens, in vivo or in vitro, are also encompassed by the invention.

The present invention provides a family of binding proteins that arecapable of binding two or more antigens, e.g., with high affinity. Inone aspect, the present invention provides an approach to construct abispecific binding protein using two parental monoclonal antibodies: mAbA, which binds to antigen A, and mAb B, which binds to antigen B. Thebinding proteins disclosed herein, in one embodiment, are capable ofbinding antigens, cytokines, chemokines, cytokine receptors, chemokinereceptors, cytokine- or chemokine-related molecules, or cell surfaceproteins.

Thus, in one aspect, binding proteins capable of binding two or moreantigens are provided. In one embodiment, the present invention providesa binding protein comprising at least two polypeptide chains, whereinthe polypeptide chains pair to form IgG-like molecules capable ofbinding two or more antigens. In one embodiment, the binding proteincomprises two, three, four, five, or more polypeptide chains. In oneembodiment, the binding protein comprises at least one VL_(A), at leastone VL_(B), at least one VH_(A), at least one VH_(B), at least one CL,and at least one CH1, wherein VL is a light chain variable domain, VH isa heavy chain variable domain, CL is a light chain constant domain, CH1is the first constant domain of the heavy chain, A is a first antigen,and B is a second antigen. In a further embodiment, the firstpolypeptide chain comprises a VL_(A), a CL, a VH_(B), and a CH1. In afurther embodiment, the binding protein further comprises an Fc. Inanother embodiment, the Fc region is a variant Fc region. In a furtherembodiment, the variant Fc region exhibits modified effector function,such as ADCC or CDC. In another embodiment, the variant Fc regionexhibits modified affinity or avidity for one or more FcγR.

In one embodiment, the binding protein comprises three polypeptidechains, wherein the first polypeptide chain comprises a VL_(A), a CL, aVH_(B), and a CH1, the second polypeptide chain comprises VH_(A) andCH1, and the third polypeptide chain comprises VL_(B) and CL. In afurther embodiment, the first polypeptide chain of the binding proteinfurther comprises an Fc. In another embodiment, the binding proteincomprises two polypeptide chains, wherein the first polypeptide chaincomprises a VL_(A), a CL, a VH_(B), and a CH1, the second polypeptidechain comprises VH_(A), CH1, VL_(B), and CL. In a further embodiment,the first polypeptide chain further comprises an Fc.

In one embodiment, the binding protein comprises three polypeptidechains, and their corresponding cDNA during co-transfection are presentat a molar ratio of first:second:third of 1:1:1, 1:1.5:1, 1:3:1,1:1:1.5, 1:1:3, 1:1.5:1.5, 1:3:1.5, 1:1.5:3, or 1:3:3. In anotherembodiment, the binding protein comprises two polypeptide chains, andtheir corresponding cDNA during co-transfection are present at a molarratio of first:second of 1:1, 1:1.5, or 1:3, or any other ratios,through optimization, in an effort to maximize the monomeric FIT-Igfraction in any given transfection.

In one embodiment, the binding protein of the present invention does notcomprise a peptide linker. In one embodiment, the binding protein of thepresent invention comprises at least one amino acid or polypeptidelinker. In a further embodiment, the linker is selected from the groupconsisting of G, GS, SG, GGS, GSG, SGG, GGG, GGGS, SGGG, GGGGS, GGGGSGS,GGGGSGS, GGGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, AKTTPKLEEGEFSEAR,AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP,SAKTTPKLGG, RADAAP, RADAAPTVS, RADAAAAGGPGS, RADAAAA(G₄S)₄, SAKTTP,SAKTTPKLGG, SAKTTPKLEEGEFSEARV, ADAAP, ADAAPTVSIFPP, TVAAP,TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP, AKTTPPSVTPLAP, AKTTAP,AKTTAPSVYPLAP, ASTKGP, ASTKGPSVFPLAP, GENKVEYAPALMALS, GPAKELTPLKEAKVS,and GHEAAAVMQVQYPAS. The linkers can also be in vivo cleavable peptidelinkers, protease (such as MMPs) sensitive linkers, disulfide bond-basedlinkers that can be cleaved by reduction, etc., as previously described(Fusion Protein Technologies for Biopharmaceuticals: Applications andChallenges, edited by Stefan R. Schmidt), or any cleavable linkers knownin the art. Such cleavable linkers can be used to release the top Fab invivo for various purposes, in order to improve tissue/cell penetrationand distribution, to enhance binding to targets, to reduce potentialside effect, as well as to modulate in vivo functional and physicalhalf-life of the 2 different Fab regions.

In one embodiment, the binding protein comprises a first polypeptidecomprising, from amino to carboxyl terminus, VL_(A)-CL-VH_(B)-CH1-Fc, asecond polypeptide chain comprising, from amino to carboxyl terminus,VH_(A)-CH1, and a third polypeptide chain comprising, from amino tocarboxyl terminus, VL_(B)-CL; wherein VL is a light chain variabledomain, CL is a light chain constant domain, VH is a heavy chainvariable domain, CH1 is the first constant domain of the heavy chain, Ais a first epitope or antigen, and B is a second epitope or antigen. Inone embodiment, the Fc region is human IgG1. In another embodiment, theFc region is a variant Fc region. In a further embodiment, the aminoacid sequence of the Fc region is at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 20. In a further embodiment, the CLof the first polypeptide chain is fused directly to VH_(B). In anotherembodiment, the CL of the first polypeptide chain is linked to VH_(B)via an amino acid or an oligopeptide linker. In a further embodiment,the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

In another embodiment, the binding protein comprises a first polypeptidecomprising, from amino to carboxyl terminus, VH_(B)-CH1-VL_(A)-CL-Fc, asecond polypeptide chain comprising, from amino to carboxyl terminus,VH_(A)-CH1, and a third polypeptide chain comprising, from amino tocarboxyl terminus, VL_(B)-CL; wherein VL is a light chain variabledomain, CL is a light chain constant domain, VH is a heavy chainvariable domain, CH1 is the first constant domain of the heavy chain, Ais a first epitope or antigen, and B is a second epitope or antigen. Inone embodiment, the Fc region is human IgG1. In another embodiment, theFc region is a variant Fc region. In a further embodiment, the aminoacid sequence of the Fc region is at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 20. In one embodiment, the CH1 ofthe first polypeptide chain is fused directly to VL_(A). In anotherembodiment, the CH1 of the first polypeptide chain is linked to VL_(A)via an amino acid or an oligopeptide linker. In a further embodiment,the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

In another embodiment, the binding protein comprises a first polypeptidecomprising, from amino to carboxyl terminus, VL_(A)-CL-VH_(B)-CH1-Fc,and a second polypeptide chain comprising, from amino to carboxylterminus, VH_(A)-CH1-VL_(B)-CL; wherein VL is a light chain variabledomain, CL is a light chain constant domain, VH is a heavy chainvariable domain, CH1 is the first constant domain of the heavy chain, Ais a first epitope or antigen, and B is a second epitope or antigen. Inone embodiment, the Fc region is human IgG1. In another embodiment, theFc region is a variant Fc region. In a further embodiment, the aminoacid sequence of the Fc region is at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 20. In a further embodiment, the CLof the first polypeptide chain is fused directly to VH_(B). In anotherembodiment, the CL of the first polypeptide chain is linked to VH_(B)via an amino acid or an oligopeptide linker. In a further embodiment,the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

In another embodiment, binding protein comprises a first polypeptidecomprising, from amino to carboxyl terminus, VH_(B)-CH1-VL_(A)-CL-Fc,and a second polypeptide chain comprising, from amino to carboxylterminus, VL_(B)-CL-VH_(A)-CH1; wherein VL is a light chain variabledomain, CL is a light chain constant domain, VH is a heavy chainvariable domain, CH1 is the first constant domain of the heavy chain, Ais a first epitope or antigen, and B is a second epitope or antigen. Inone embodiment, the Fc region is human IgG1. In another embodiment, theFc region is a variant Fc region. In a further embodiment, the aminoacid sequence of the Fc region is at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 20. In one embodiment, the CH1 ofthe first polypeptide chain is fused directly to VL_(A). In anotherembodiment, the CH1 of the first polypeptide chain is linked to VL_(A)via an amino acid or an oligopeptide linker. In a further embodiment,the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

The binding proteins of the present invention are capable of bindingpairs of cytokines. For example, the binding proteins of the presentinvention are capable of binding pairs of cytokines selected from thegroup consisting of IL-1α and IL-1β; IL-12 and IL-18, TNFα and IL-23,TNFα and IL-13; TNF and IL-18; TNF and IL-12; TNF and IL-1beta; TNF andMIF; TNF and IL-6, TNF and IL-6 Receptor, TNF and IL-17; IL-17 andIL-20; IL-17 and IL-23; TNF and IL-15; TNF and VEGF; VEGFR and EGFR;PDGFR and VEGF, IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 andTGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13and SPRR2b; IL-13 and ADAM8; and TNFα and PGE4, IL-13 and PED2, TNF andPEG2. In one embodiment, the binding proteins of the present inventionare capable of binding IL-17 and IL-20. The binding proteins of thepresent invention, in one embodiment, are capable of binding IL-17 andIL-20 and comprise variable heavy and light chains derived from theanti-IL-17 antibody LY and the anti-IL-20 antibody 15D2. In oneembodiment, the binding proteins of the present invention are capable ofbinding IL-17 and TNF. The binding proteins of the present invention, inone embodiment, are capable of binding IL-17 and TNF and comprisevariable heavy and light chains derived from the anti-IL-17 antibody LYand the TNF antibody golimumab.

In one embodiment, the binding proteins of the present invention bindIL-17 and IL-20 and comprise a first polypeptide comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 15, 25, and 27; a second polypeptidechain comprising, consisting essentially of, or consisting of an aminoacid sequence according to SEQ ID NO: 21; and a third polypeptide chaincomprising, consisting essentially of, or consisting of a sequenceaccording to SEQ ID NO: 23. In another embodiment, the binding proteinsof the present invention bind IL-27 and IL-20 and comprise a firstpolypeptide chain comprising, consisting essentially of, or consistingof an amino acid sequence selected from the group consisting of SEQ IDNOs: 15, 25, and 27, and a second polypeptide chain comprising,consisting essentially of, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 29, 30, and 31.

In one embodiment, the binding proteins of the present invention bindTNF and IL-17 and comprise a first polypeptide comprising, consistingessentially of, or consisting of an amino acid sequence according to SEQID NOs: 87; a second polypeptide chain comprising, consistingessentially of, or consisting of an amino acid sequence according to SEQID NO: 89; and a third polypeptide chain comprising, consistingessentially of, or consisting of a sequence according to SEQ ID NO: 91.

In another embodiment, the binding protein is capable of binding pairsof targets selected from the group consisting of CD137 and CD20, CD137and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 and PDL-1, VEGF andPD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1,EGFR and DLL-4, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 andCD3; CD3 and CD33; CD3 and CD133; CD47 and CD20, CD38 and CD138; CD38and CD20; CD20 and CD22; CD38 and CD40; CD40 and CD20; CD-8 and IL-6;CSPGs and RGM A; CTLA-4 and BTNO2; IGF1 and IGF2; IGF1/2 and Erb2B;IGF-1R and EGFR; EGFR and CD13; IGF-1R and ErbB3; EGFR-2 and IGFR;VEGFR-2 and Met; VEGF-A and Angiopoietin-2 (Ang-2); IL-12 and TWEAK;IL-13 and IL-1beta; PDGFR and VEGF, EpCAM and CD3, Her2 and CD3, CD19and CD3, EGFR and Her3, CD16a and CD30, CD30 and PSMA, EGFR and CD3, CEAand CD3, TROP-2 and HSG, TROP-2 and CD3, MAG and RGM A; NgR and RGM A;NogoA and RGM A; OMGp and RGM A; PDL-1 and CTLA-4; CTLA-4 and PD-1; PD-1and TIM-3; RGM A and RGM B; Te38 and TNFα; TNFα and Blys; TNFα andCD-22; TNFα and CTLA-4 domain; TNFα and GP130; TNFα and IL-12p40; andTNFα and RANK ligand, Factor IXa, Factor X. In one embodiment, thebinding proteins of the present invention are capable of binding CD3 andCD20. The binding proteins of the present invention, in one embodiment,are capable of binding CD3 and CD20 and comprise variable heavy andlight chains derived from the anti-CD3 antibody OKT3 and the anti-CD20antibody ofatumumab. In one embodiment, the binding proteins of thepresent invention are capable of binding CTLA-4 and PD-1. The bindingproteins of the present invention, in one embodiment, are capable ofbinding CTLA-4 and PD-1 and comprise variable heavy and light chainsderived from the CTLA-4 antibody ipilimumab and the PD-1 antibodynivolumab.

In one embodiment, the binding proteins of the present invention bindCD3 and CD20 and comprise a first polypeptide chain comprising,consisting essentially of, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 41 and 48; a secondpolypeptide chain comprising, consisting essentially of, or consistingof an amino acid sequence according to SEQ ID NO: 44; and a thirdpolypeptide chain comprising, consisting essentially of, or consistingof an amino acid sequence according to SEQ ID NO: 46.

In one embodiment, the binding proteins of the present invention bindCTLA-4 and PD-1 and comprise a first polypeptide chain comprising,consisting essentially of, or consisting of an amino acid sequenceaccording to SEQ ID NO: 92; a second polypeptide chain comprising,consisting essentially of, or consisting of an amino acid sequenceaccording to SEQ ID NO: 95; and a third polypeptide chain comprising,consisting essentially of, or consisting of an amino acid sequenceaccording to SEQ ID NO: 97.

In one embodiment, the binding protein provided herein is capable ofbinding one or more epitopes on CTLA-4. In one embodiment, the bindingprotein provided herein is capable of binding one or more peptides onPD-1.

In one embodiment, the binding protein is capable of binding one or moreepitopes on one or more immune checkpoint protein on T cells such as,for example, TIM-3, Lag3, ICOS, BTLA, CD160, 2B4, KIR, CD137, CD27,OX40, CD40L, and A2aR. In another embodiment, the binding protein iscapable of binding one or more epitopes on one or more tumor cellsurface protein that is involved with immune checkpoint pathways, suchas, for example, PD-L1, PD-L2, Galectin9, HVEM, CD48, B7-1, B7-2, ICOSL,B7-H3, B7-H4, CD137L, OX40L, CD70, and CD40.

In one aspect, the present invention provides pharmaceuticalcompositions comprising the binding proteins described herein. In oneembodiment, provided herein are pharmaceutical compositions comprisingthe binding protein of any one of the preceding claims and one or morepharmaceutically acceptable carrier.

In another aspect, the present invention provides methods of treating orpreventing an inflammatory disease, autoimmune disease,neurodegenerative disease, cancer, sepsis, or spinal cord injury in asubject in need thereof. In one embodiment, the method comprisesadministering to a subject an effective amount of one or more of thebinding proteins provided herein, or one or more pharmaceuticalcompositions comprising the binding proteins provided herein and apharmaceutically acceptable carrier. Uses of the binding proteinsdescribed herein in the manufacture of a medicament for treatment orprevention of an inflammatory disease, autoimmune disease,neurodegenerative disease, cancer, or spinal cord injury are alsoprovided herein. In one embodiment, the inflammatory disease, autoimmunedisease, or neurodegenerative disease is selected from the groupconsisting of asthma, rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis, Alzheimer's disease, or Parkinson'sdisease.

In one embodiment, the present disclosure provides methods for treatingor preventing rheumatoid arthritis, psoriasis, osteoporosis, stroke,liver disease, or oral cancer to a subject in need thereof, the methodcomprising administering to the subject a FIT-Ig binding proteindescribed herein, wherein the binding protein is capable of bindingIL-17 and IL-20. In a further embodiment, the FIT-Ig binding proteincomprises an amino acid sequence selected from SEQ ID NOs: 15, 25, and27; and amino acid sequence according to SEQ ID NO: 21; and an aminoacid sequence according to SEQ ID NO: 23. In another embodiment, theFIT-Ig binding protein comprises an amino acid sequence selected fromSEQ ID NOs: 15, 25, and 27; and an amino acid sequence selected from SEQID NOs: 29, 30 and 31.

In one embodiment, the present disclosure provides methods for treatingor preventing a B cell cancer in a subject in need thereof, the methodcomprising administering to the subject a FIT-Ig binding protein,wherein the FIT-Ig binding protein is capable of binding one or more Bcell antigen. In a further embodiment, the FIT-Ig binding protein iscapable of binding CD20. In a further embodiment, the FIT-Ig bindingprotein is capable of binding CD20 and another antigen. In a furtherembodiment, the binding protein is capable of binding CD3 and CD20. In afurther embodiment, the cancer is a B cell cancer. In a still furtherembodiment, the B cell cancer is selected from the group consisting ofHodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], precursor B celllymphoblastic leukemia/lymphoma, mature B cell neoplasms, B cell chroniclymphocytic leukemia/small lymphocytic lymphoma, B cell prolymphocyticleukemia, lymphoplamacytic lymphoma, mantle cell lymphoma, follicularlymphoma, cutaneous follicle center lymphoma, marginal zone B celllymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt'slymphoma, plasmacytoma, plasma cell myeloma, post-transplantlymphoproliferative disorder, Waldenstrom's macroglobulinemia, andanaplastic large-cell lymphoma. In one embodiment, the presentdisclosure provides methods for treating or preventing a B cell cancerin a subject in need thereof, the method comprising administering to thesubject a FIT-Ig binding protein, wherein the FIT-Ig binding proteincomprises an amino acid sequence according to SEQ ID NOs: 41 or 48; andamino acid sequence according to SEQ ID NO: 44, and an amino acidsequence according to SEQ ID NO: 46.

In one embodiment, the present disclosure provides methods for treatingor preventing an autoimmune disease, inflammatory disease, or infectionin a subject in need thereof, the method comprising administering to thesubject a FIT-Ig binding protein described herein, wherein the bindingprotein is capable of binding TNF and IL-17. In a further embodiment,the FIT-Ig binding protein comprises sequences according to SEQ ID NOs:87, 89, and 91. In another embodiment, the present disclosure providesmethods for treating or preventing an autoimmune or inflammatorydisease, the method comprising administering to the subject a FIT-Igbinding protein, wherein the binding protein is capable of binding TNFand IL-17, and wherein the autoimmune or inflammatory disease isselected from the group consisting of Crohn's disease, psoriasis(including plaque psoriasis), arthritis (including rheumatoid arthritis,psoriatic arthritis, osteoarthritis, or juvenile idiopathic arthritis),multiple sclerosis, ankylosing spondylitis, spondylosing arthropathy,systemic lupus erythematosus, uveitis, sepsis, neurodegenerativediseases, neuronal regeneration, spinal cord injury, primary andmetastatic cancers, a respiratory disorder; asthma; allergic andnonallergic asthma; asthma due to infection; asthma due to infectionwith respiratory syncytial virus (RSV); chronic obstructive pulmonarydisease (COPD); a condition involving airway inflammation; eosinophilia;fibrosis and excess mucus production; cystic fibrosis; pulmonaryfibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema;allergic rhinitis; allergic enterogastritis; an inflammatory and/orautoimmune condition of the skin; an inflammatory and/or autoimmunecondition of gastrointestinal organs; inflammatory bowel diseases (IBD);ulcerative colitis; an inflammatory and/or autoimmune condition of theliver; liver cirrhosis; liver fibrosis; and liver fibrosis caused byhepatitis B and/or C virus; scleroderma. In another embodiment, Inanother embodiment, the present disclosure provides methods for treatingor preventing cancer in a subject in need thereof, the method comprisingadministering to the subject a FIT-Ig binding protein described herein,wherein the binding protein is capable of binding TNF and IL-17. In afurther embodiment, the cancer is hepatocellular carcinoma;glioblastoma; lymphoma; or Hodgkin's lymphoma. In another embodiment,the present disclosure provides methods for treating or preventing andinfection in a subject in need thereof, the method comprisingadministering to the subject a FIT-Ig binding protein described herein,wherein the infection is a viral infection, a bacterial infection, aparasitic infection, HTLV-1 infection. In one embodiment, the presentdisclosure provides methods for suppression of expression of protectivetype 1 immune responses, and suppression of expression of a protectivetype 1 immune response during vaccination.

In one embodiment, the present disclosure provides methods for treatingrheumatoid arthritis in a subject in need thereof, the method comprisingadministering to the subject a FIT-Ig binding protein, wherein thebinding protein comprises sequences according to SEQ ID NOs: 87, 89, and91.

In one embodiment, the present disclosure provides methods for treatingor preventing cancer in a subject in need thereof, the method comprisingadministering to the subject a FIT-Ig binding protein described herein,wherein the binding protein is capable of binding CTLA-4 and PD-1. In afurther embodiment, the FIT-Ig binding protein comprises an amino acidsequence comprising SEQ ID NOs: 92, 95, and 97. In another embodiment,the present disclosure provides methods for treating or preventingcancer in a subject in need thereof, wherein the binding protein iscapable of binding CTLA-4 and PD-1, and wherein the cancer is a cancertypically responsive to immunotherapy. In another embodiment, the canceris a cancer that has not been associated with immunotherapy. In anotherembodiment, the cancer is a cancer that is a refractory or recurringmalignancy. In another embodiment, the binding protein inhibits thegrowth or survival of tumor cells. In another embodiment, the cancer isselected from the group consisting of melanoma (e.g., metastaticmalignant melanoma), renal cancer (e.g. clear cell carcinoma), prostatecancer (e.g. hormone refractory prostate adenocarcinoma), pancreaticadenocarcinoma, breast cancer, colon cancer, lung cancer (e.g. non-smallcell lung cancer), esophageal cancer, squamous cell carcinoma of thehead and neck, liver cancer, ovarian cancer, cervical cancer, thyroidcancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplasticmalignancies.

In one embodiment, the present disclosure provides methods for treatingor preventing melanoma in a subject in need thereof, the methodcomprising administering to the subject a FIT-Ig binding proteindescribed herein, wherein the binding protein is capable of bindingCTLA-4 and PD-1. In a further embodiment, the present disclosureprovides methods for treating or preventing melanoma in a subject inneed thereof, wherein the method comprises administering to the subjecta FIT-Ig binding protein comprising amino acid sequences according toSEQ ID NOs: 92, 95, and 97.

In another embodiment, the present disclosure provides methods fortreating or preventing infections or infectious disease in a subject inneed thereof, the method comprising administering to the subject aFIT-Ig binding protein described herein, wherein the binding protein iscapable of binding CTLA-4 and PD-1. In one embodiment, the FIT-Igbinding protein is administered alone, or in combination with vaccines,to stimulate the immune response to pathogens, toxins, andself-antigens. Therefore, in one embodiment, the binding proteinsprovided herein can be used to stimulate immune response to virusesinfectious to humans, such as, but not limited to, humanimmunodeficiency viruses, hepatitis viruses class A, B and C, EpsteinBarr virus, human cytomegalovirus, human papilloma viruses, herpesviruses, bacteria, fungal parasites, or other pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of FIT-Igs that are made up of threeconstructs, such as FIT1-Ig, FIT2-Ig, and FIT3-Ig. FIG. 1B shows thethree constructs used to prepare such FIT1-Igs.

FIG. 2A shows the basic structure of FIT-Igs that are made up of twoconstructs. FIG. 2B shows the two constructs used to prepare suchFIT-Igs.

FIG. 3 provides the dual-specific antigen binding of FIT1-Ig as measuredby Biacore. The top panel of FIG. 3 shows the results of the Biacorebinding assay in which FIT1-Ig was first saturated by IL-17, followed byIL-20. The bottom panel of FIG. 3 shows the results of the Biacore assayin which FIT1-Ig was first saturated by IL-20, followed by IL-17.

FIG. 4. Shows the solubility at a range of pH of anti-IL-17/IL-20 FIT Igor monoclonal antibody rituximab, as measured by PEG-inducedprecipitation.

FIG. 5 shows the stability of stable CHO cell line development in bothDG44 (5A and 5B) and CHO-S (5C and 5D) systems.

FIG. 6 shows the binding to CTLA-4 (6A) or PD-1 (FIG. 6B) by FIT10-Ig orthe parental antibodies Ipilimumab and Nivolumab, as assessed by ELISA.

FIG. 7 shows a multiple binding study of FIT10-Ig against both CTLA-4and PD-1. Binding to CTLA-4 followed by PD-1; and binding by PD-1followed by CTLA-4 are both shown as indicated in FIG. 7.

DETAILED DESCRIPTION

The present invention relates to multivalent and multispecific bindingproteins, methods of making the binding proteins, and to their uses inthe prevention and/or treatment of acute and chronic inflammatorydiseases and disorders, cancers, and other diseases. This inventionpertains to multivalent and/or multispecific binding proteins capable ofbinding two or more antigens. Specifically, the invention relates toFabs-in-tandem immunoglobulins (FIT-Ig), and pharmaceutical compositionsthereof, as well as nucleic acids, recombinant expression vectors andhost cells for making such FIT-Igs. Methods of using the FIT-Igs of theinvention to detect specific antigens, either in vitro or in vivo arealso encompassed by the invention.

The novel family of binding proteins provided herein are capable ofbinding two or more antigens, e.g., with high affinity. Specifically,the present invention provides an approach to construct a bispecificbinding protein using 2 parental monoclonal antibodies: mAb A, whichbinds to antigen a; and mAb B, which binds to antigen b.

In one aspect, the present invention provides a binding proteincomprising a variable light chain specific for a first antigen orepitope, a first light chain constant domain, a variable heavy chainspecific for a second antigen or epitope, a first heavy chain CH1, avariable heavy chain specific for the first antigen or epitope, a secondheavy chain CH1, a variable heavy chain specific for the second antigenor epitope, and a second light chain constant domain. In one embodiment,the binding protein further comprises an Fc region. The binding proteinmay further comprise one or more amino acid or polypeptide linkerlinking two or more of the components of the binding protein. Forexample, the binding protein may comprise a polypeptide linker linkingthe light chain variable region to the light chain constant region.

In one embodiment, the present disclosure provides a binding proteincomprising a polypeptide chain comprisingVL_(A)-CL-(X1)n-VH_(B)-CH1-(X2)n, wherein VLA is the light chainvariable domain of mAb A, CL is a light chain constant domain, X1represents an amino acid or an oligopeptide linker, VH_(B) is the heavychain variable domain of mAb B, CH1 is the first constant domain of theheavy chain, X2 represents an Fc region or a different dimerizationdomain, and n is 0 or 1.

In one embodiment, the invention provides a binding protein comprisingthree different polypeptide chains (FIG. 1), wherein the firstpolypeptide chain (construct #1) comprisesVL_(A)-CL-(X1)n-VH_(B)-CH1-(X2)n, wherein VLA is the light chainvariable domain of mAb A, CL is a light chain constant domain, X1represents an amino acid or an oligopeptide linker, VH_(B) is the heavychain variable domain of mAb B, CH1 is the first constant domain of theheavy chain, X2 represents an Fc region or a different dimerizationdomain, and n is 0 or 1. The second polypeptide chain (construct #2)comprises VH_(A)-CH1, wherein VH_(A) is the heavy chain variable domainof mAb A, and CH1 is the first constant domain of the heavy chain. Thethird polypeptide chain (construct #3) comprises VL_(B)-CL, whereinVL_(B) is the light chain variable domain of mAb B, and CL is theconstant domain of the light chain.

In another embodiment, the invention provides a binding proteincomprising three different polypeptide chains with the overall moleculardesign similar to the previous embodiment except the order of thevariable domains are reversed. In the embodiment the first polypeptidechain comprises VH_(B)-CH1-(X1)n-VL_(A)-CL-(X2)n, wherein VL_(A) is alight chain variable domain of mAb A, CL is a light chain constantdomain, X1 represents an amino acid or an oligopeptide linker, VH_(B) isthe heavy chain variable domain of mAb B, CH1 is the first constantdomain of the heavy chain, X2 represents an Fc region or a differentdimerization domain, and n is 0 or 1. The second polypeptide chaincomprises VH_(A)-CH1, wherein VH_(A) is the heavy chain variable domainof mAb A and CH1 is the first constant domain of the heavy chain. Thethird polypeptide chain comprises VL_(B)-CL, wherein VL_(B) is the lightchain variable domain of mAb B and CL is the constant domain of thelight chain.

In another embodiment the invention provides a binding proteincomprising two different polypeptide chains (FIG. 2), wherein the firstpolypeptide chain (construct #1) comprisesVL_(A)-CL-(X1)n-VH_(B)-CH1-(X2)n, wherein VL_(A) is a light chainvariable domain of mAb A, CL is a light chain constant domain, X1represents an amino acid or an oligopeptide linker, VH_(B) is the heavychain variable domain of mAb B, CH1 is the first constant domain of theheavy chain, X2 represents an Fc region or a different dimerizationdomain, and n is 0 or 1. The second polypeptide chain (construct #4)comprises VH_(A)-CH1-(X3)n-VL_(B)-CL, wherein VH_(A) is the heavy chainvariable domain of mAb A, CH1 is the first constant domain of the heavychain, X3 represents an amino acid or polypeptide that is not a constantdomain, n is 0 or 1, VL_(B) is the light chain variable domain of mAb B,and CL is the constant domain of the light chain.

In another embodiment the invention provides a binding proteincomprising two polypeptide chains with the overall molecular designsimilar to the previous embodiment except the order of the variabledomains are reversed. In this embodiment the first polypeptide chaincomprises VH_(B)-CH1-(X1)n-VL_(A)-CL-(X2)n, wherein VL_(A) is a lightchain variable domain of mAb A, CL is a light chain constant domain, X1represents an amino acid or an oligopeptide linker, VH_(B) is the heavychain variable domain of mAb B, CH1 is the first constant domain of theheavy chain, X2 represents an Fc region or a different dimerizationdomain, and n is 0 or 1. The second polypeptide chain comprisesVL_(B)-CL-(X3)n-VH_(A)-CH1, wherein VH_(A) is the heavy chain variabledomain of mAb A, CH1 is the first constant domain of the heavy chain, X3represents an amino acid or an oligopeptide linker, n is 0 or 1, VL_(B)is the light chain variable domain of mAb B, and CL is the constantdomain of the light chain.

In one embodiment, the VH and VL domains in the binding protein areselected from the group consisting of murine heavy/light chain variabledomains, fully human heavy/light chain variable domains, CDR graftedheavy/light chain variable domains, humanized heavy/light chain variabledomains, and mixtures thereof. In a preferred embodiment VH_(A)/VL_(A)and VH_(B)/VL_(B) are capable of binding the same antigen. In anotherembodiment VH_(A)/VL_(A) and VH_(B)/VL_(B) are capable of bindingdifferent antigens.

In one embodiment, the first polypeptide chain comprisesVL_(A)-CL-VH_(B)-CH1-Fc, and the CL and VH_(B) of the first polypeptidechain are directly fused together. In another embodiment, the CL andVH_(B) are linked by an amino acid or an oligopeptide linker. In anotherembodiment, the first polypeptide chain comprisesVH_(B)-CH1-VL_(A)-CL-Fc, and the CH1 and VL_(A) are directly fusedtogether. In another embodiment, the CH1 and VL_(A) are linked by anamino acid or an oligopeptide linker. In a further embodiment, theoligo- or poly-peptide linker comprises 1 or more amino acids of anyreasonable sequence that provides flexibility. Preferably the linker isselected from the group consisting of G, GS, SG, GGS, GSG, SGG, GGG,GGGS, SGGG, GGGGS, GGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGS,GGGGSGGGGSGGGGS, AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, AKTTPKLGG,SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RADAAP, RADAAPTVS,RADAAAAGGPGS, RADAAAA(G₄S)₄, SAKTTP, SAKTTPKLGG, SAKTTPKLEEGEFSEARV,ADAAP, ADAAPTVSIFPP, TVAAP, TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP,AKTTPPSVTPLAP, AKTTAP, AKTTAPSVYPLAP, ASTKGP, ASTKGPSVFPLAP,GENKVEYAPALMALS, GPAKELTPLKEAKVS, and GHEAAAVMQVQYPAS. In oneembodiment, the amino acid sequence of the linker may be selected fromthe group consisting of SEQ ID NOs. 26, 28, and 49-86. In oneembodiment, the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO:28). The linkers can also be in vivo cleavable peptide linkers, protease(such as MMPs) sensitive linkers, disulfide bond-based linkers that canbe cleaved by reduction, etc., as previously described (Fusion ProteinTechnologies for Biopharmaceuticals: Applications and Challenges, editedby Stefan R. Schmidt), or any cleavable linkers known in the art. Suchcleavable linkers can be used to release the top Fab in vivo for variouspurposes, in order to improve tissue/cell penetration and distribution,to enhance binding to targets, to reduce potential side effect, as wellas to modulate in vivo functional and physical half-life of the 2different Fab regions. In one embodiment, the binding protein comprisesan Fc region. As used herein, the term “Fc region” refers to theC-terminal region of an IgG heavy chain. An example of the amino acidsequence containing the human IgG1 Fc region is SEQ ID NO: 20. The Fcregion of an IgG comprises two constant domains, CH2 and CH3.

In one embodiment, the Fc region is a variant Fc region. In oneembodiment, the variant Fc region has one or more amino acidmodifications, such as substitutions, deletions, or insertions, relativeto the parent Fc region. In a further embodiment, amino acidmodifications of the Fc region alter the effector function activityrelative to the parent Fc region activity. For example, in oneembodiment, the variant Fc region may have altered (i.e., increased ordecreased) antibody-dependent cytotoxicity (ADCC), complement-mediatedcytotoxicity (CDC), phagocytosis, opsonization, or cell binding. Inanother embodiment, amino acid modifications of the Fc region may alter(i.e., increase or decrease) the affinity of the variant Fc region foran FcγR relative to the parent Fc region. For example, the variant Fcregion may alter the affinity for FcγRI, FcγRII, FcγRIII.

In one preferred embodiment, the binding proteins provided herein arecapable of binding one or more targets. In one embodiment, the target isselected from the group consisting of cytokines, cell surface proteins,enzymes and receptors. Preferably the binding protein is capable ofmodulating a biological function of one or more targets. More preferablythe binding protein is capable of neutralizing one or more targets.

In one embodiment, the binding protein of the invention is capable ofbinding cytokines selected from the group consisting of lymphokines,monokines, and polypeptide hormones. In a further embodiment, thebinding protein is capable of binding pairs of cytokines selected fromthe group consisting of IL-1α and IL-1β; IL-12 and IL-18, TNFα andIL-23, TNFα and IL-13; TNF and IL-18; TNF and IL-12; TNF and IL-1beta;TNF and MIF; TNF and IL-6, TNF and IL-6 Receptor, TNF and IL-17; IL-17and IL-20; IL-17 and IL-23; TNF and IL-15; TNF and VEGF; VEGFR and EGFR;IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHRagonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 andADAM8; and TNFα and PGE4, IL-13 and PED2, TNF and PEG2.

In another embodiment, the binding protein of the invention is capableof binding pairs of targets selected from the group consisting of CD137and CD20, CD137 and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 andPDL-1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1,TIM-3 and PDL-1, EGFR and DLL-4, VEGF and EGFR, HGF and VEGF, VEGF andVEGF (same or a different epitope), VEGF and Ang2, EGFR and cMet, PDGFand VEGF, VEGF and DLL-4, OX40 and PD-L1, ICOS and PD-1, ICOS and PD-L1,Lag-3 and PD-1, Lag-3 and PD-L1, Lag-3 and CTLA-4, ICOS and CTLA-4,CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3 andCD33; CD3 and CD133; CD38 & CD138; CD38 and CD20; CD20 and CD22; CD38and CD40; CD40 and CD20; CD47 and CD20, CD-8 and IL-6; CSPGs and RGM A;CTLA-4 and BTNO2; CTLA-4 and PD-1; IGF1 and IGF2; IGF1/2 and Erb2B;IGF-1R and EGFR; EGFR and CD13; IGF-1R and ErbB3; EGFR-2 and IGFR; Her2and Her2 (same or a different epitope); Factor IXa, Factor X, VEGFR-2and Met; VEGF-A and Angiopoietin-2 (Ang-2); IL-12 and TWEAK; IL-13 andIL-1beta; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A;PDL-1 and CTLA-4; PD-1 and TIM-3; RGM A and RGM B; Te38 and TNFα; TNFαand Blys; TNFα and CD-22; TNFα and CTLA-4 domain; TNFα and GP130; TNFαand IL-12p40; and TNFα and RANK ligand.

In one embodiment, the binding protein is capable of binding human IL-17and human IL-20. In a further embodiment, the binding protein is capableof binding human IL-17 and human IL-20 and comprises a FIT-Igpolypeptide chain #1 sequence that is about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identicalto a sequence selected from the group consisting of SEQ ID NO. 15, 25,and 27; a polypeptide chain #2 sequence that is about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or100% identical to SEQ ID NO. 21; and a polypeptide chain #3 sequencethat is about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 23. Inanother embodiment, the binding protein is capable of binding humanIL-17 and human IL-20 and comprises FIT-Ig polypeptide chain #1 sequencethat is about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99%, or 100% identical to a sequence selected fromthe group consisting of SEQ ID NO. 15, 25, and 27; and a polypeptidechain #4 that is about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 99%, or 100% identical to a sequenceselected from the group consisting of SEQ ID NO. 29, 30, and 31.

In one embodiment, the binding protein is capable of binding human CD3and human CD20. In a further embodiment, the binding protein comprises aFIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%identical to a sequence selected from the group consisting of SEQ ID NO.41 and 48; a polypeptide chain #2 sequence that is about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or100% identical to SEQ ID NO. 44; and a polypeptide chain #3 sequencethat is about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 46.

In one embodiment, the binding protein is capable of binding human IL-17and human TNF. In a further embodiment, the binding protein is capableof binding human IL-17 and human TNF and comprises a FIT-Ig polypeptidechain #1 sequence that is about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ IDNO. 87; a polypeptide chain #2 sequence that is about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or100% identical to SEQ ID NO. 89; and a polypeptide chain #3 sequencethat is about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 91.

In one embodiment, the binding protein is capable of binding humanCTLA-4 and human PD-1. In a further embodiment, the binding protein iscapable of binding human CTLA-4 and human PD-1 and comprises a FIT-Igpolypeptide chain #1 sequence that is about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identicalto SEQ ID NO. 92 a polypeptide chain #2 sequence that is about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about99%, or 100% identical to SEQ ID NO. 95; and a polypeptide chain #3sequence that is about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 97.

In another embodiment, the binding protein of the invention is capableof binding one or two cytokines, cytokine-related proteins, and cytokinereceptors selected from the group consisting of BMP1, BMP2, BMP3B(GDF10), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF),EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6(HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16,FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4,IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FIL1, FIL1 (EPSILON), FIL1(ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11,IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20,IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA,FGER1, FGFR2, FGFR3, EGFR, ROR1, 2B4, KIR, CD137, CD27, OX40, CD40L,A2aR, CD48, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CD70, CD40,PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4(OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL),TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14(HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC,IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA,IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R,IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP,IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.

The binding protein of the invention is capable of binding one or morechemokines, chemokine receptors, and chemokine-related proteins selectedfrom the group consisting of CCL1 (I-309), CCL2 (MCP-1/MCAF), CCL3(MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2),CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17(TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL20 (MIP-3a), CCL21(SLC/exodus-2), CCL22 (MDC/STC-1), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK/ILC),CCL28, CXCL1 (GRO1), CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6(GCP-2), CXCL9 (MIG), CXCL10 (IP 10), CXCL11 (I-TAC), CXCL12 (SDF1),CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1),SCYE1, XCL1 (lymphotactin), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2(D6/JAB61), CCR1 (CKR1/HM145), CCR2 (mcp-1RB/RA), CCR3 (CKR3/CMKBR3),CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7(CKR7/EBI1), CCR8 (CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1),CCRL2 (L-CCR), XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28),CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6(TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R(GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7,CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A,IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

In another embodiment, the binding protein of the invention is capableof binding cell surface protein such as, for example, integrins. Inanother embodiment, the binding protein of the invention is capable ofbinding enzymes selected from the group consisting of kinases andproteases. In yet another embodiment, the binding protein of theinvention is capable of binding receptors selected from the groupconsisting of lymphokine receptors, monokine receptors, and polypeptidehormone receptors.

In one embodiment, the binding protein is multivalent. In anotherembodiment, the binding protein is multispecific. The multivalent and ormultispecific binding proteins described above have desirable propertiesparticularly from a therapeutic standpoint. For instance, themultivalent and or multispecific binding protein may (1) be internalized(and/or catabolized) faster than a bivalent antibody by a cellexpressing an antigen to which the antibodies bind; (2) be an agonistantibody; and/or (3) induce cell death and/or apoptosis of a cellexpressing an antigen which the multivalent antibody is capable ofbinding to. The “parent antibody” which provides at least one antigenbinding specificity of the multivalent and or multispecific bindingproteins may be one which is internalized (and/or catabolized) by a cellexpressing an antigen to which the antibody binds; and/or may be anagonist, cell death-inducing, and/or apoptosis-inducing antibody, andthe multivalent and or multispecific binding protein as described hereinmay display improvement(s) in one or more of these properties. Moreover,the parent antibody may lack any one or more of these properties, butmay be endowed with them when constructed as a multivalent bindingprotein as herein described.

In another embodiment the binding protein of the invention has an onrate constant (Kon) to one or more targets selected from the groupconsisting of: at least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹; atleast about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about 10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance. Preferably, thebinding protein of the invention has an on rate constant (Kon) to one ormore targets between 10²M⁻¹s⁻¹ to 10³M⁻¹s⁻¹; between 10³ M⁻¹s⁻¹ to 10⁴M⁻¹s⁻¹; between 10⁴ M⁻¹s⁻¹ to 10⁵M⁻¹s⁻¹; or between 10⁵ M⁻¹s⁻¹ to 10⁶M⁻¹s⁻¹; as measured by surface plasmon resonance.

In another embodiment the binding protein has an off rate constant(Koff) for one or more targets selected from the group consisting of: atmost about 10⁻³s⁻¹; at most about 10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹; and atmost about 10⁻⁶s⁻¹, as measured by surface plasmon resonance.Preferably, the binding protein of the invention has an off rateconstant (Koff) to one or more targets of 10⁻³s⁻¹ to 10⁻⁴s⁻¹; of 10⁻⁴s⁻¹to 10⁻⁵s⁻¹; or of 10⁻⁵s⁻¹ to 10⁻⁶s⁻¹, as measured by surface plasmonresonance.

In another embodiment the binding protein has a dissociation constant(K_(D)) to one or more targets selected from the group consisting of: atmost about 10⁻⁷ M; at most about 10⁻⁸ M; at most about 10⁻⁹ M; at mostabout 10⁻¹⁰; at most about 10⁻¹¹ M; at most about 10⁻¹² M; and at most10⁻¹³ M. Preferably, the binding protein of the invention has adissociation constant (K_(D)) to IL-12 or IL-23 of 10⁻⁷ M to 10⁻⁸ M; of10⁻⁸ M to 10⁻⁹ M; of 10⁻⁹ M to 10⁻¹⁰ M; of 10⁻¹⁰ to 10⁻¹¹ M; of 10⁻¹¹ Mto 10⁻¹² M; or of 10⁻¹² M to 10⁻¹³ M.

In another embodiment, the binding protein described above is aconjugate further comprising an agent selected from the group consistingof an immunoadhesion molecule, an imaging agent, a therapeutic agent,and a cytotoxic agent. In one embodiment, the imaging agent is selectedfrom the group consisting of a radiolabel, an enzyme, a fluorescentlabel, a luminescent label, a bioluminescent label, a magnetic label,and biotin. In a further embodiment, the imaging agent is a radiolabelselected from the group consisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. In one embodiment, the therapeuticor cytotoxic agent is selected from the group consisting of animmunosuppressive agent, an immuno-stimulatory agent, ananti-metabolite, an alkylating agent, an antibiotic, a growth factor, acytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, a toxin, and an apoptotic agent. In one embodiment, thebinding protein is conjugated directly to the agent. In anotherembodiment, the binding protein is conjugated to the agent via a linker.Suitable linkers include, but are not limited to, amino acid andpolypeptide linkers disclosed herein. Linkers may be cleavable ornon-cleavable.

In another embodiment the binding protein described above is acrystallized binding protein and exists as a crystal. Preferably thecrystal is a carrier-free pharmaceutical controlled release crystal.More preferably the crystallized binding protein has a greater half lifein vivo than the soluble counterpart of said binding protein. Mostpreferably the crystallized binding protein retains biological activity.

In another embodiment the binding protein described above isglycosylated. Preferably, the glycosylation is a human glycosylationpattern.

One aspect of the invention pertains to an isolated nucleic acidencoding any one of the binding protein disclosed above. A furtherembodiment provides a vector comprising the isolated nucleic aciddisclosed above wherein said vector is selected from the groupconsisting of pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002,Vol 30, No. 2); pTT3 (pTT with additional multiple cloning site; pEFBOS(Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol 18, No.17); pBV; pJV; pcDNA3.1 TOPO, pEF6 TOPO and pBJ. The multi-specificbinding proteins and methods of making the same are provided. Thebinding protein can be generated using various techniques. Expressionvectors, host cells and methods of generating the binding proteins areprovided in this disclosure.

The antigen-binding variable domains of the binding proteins of thisdisclosure can be obtained from parent binding proteins, includingpolyclonal Abs, monoclonal Abs, and or receptors capable of bindingantigens of interest. These parent binding proteins may be naturallyoccurring or may be generated by recombinant technology. The person ofordinary skill in the art is well familiar with many methods forproducing antibodies and/or isolated receptors, including, but notlimited to using hybridoma techniques, selected lymphocyte antibodymethod (SLAM), use of a phage, yeast, or RNA-protein fusion display orother library, immunizing a non-human animal comprising at least some ofthe human immunoglobulin locus, and preparation of chimeric,CDR-grafted, and humanized antibodies. See, e.g., US Patent PublicationNo. 20090311253 A1. Variable domains may also be prepared using affinitymaturation techniques. The binding variable domains of the bindingproteins can also be obtained from isolated receptor molecules obtainedby extraction procedures known in the art (e.g., using solvents,detergents, and/or affinity purifications), or determined by biophysicalmethods known in the art (e.g., X-ray crystallography, NMR,interferometry, and/or computer modeling).

An embodiment is provided comprising selecting parent binding proteinswith at least one or more properties desired in the binding proteinmolecule. In an embodiment, the desired property is one or more of thoseused to characterize antibody parameters, such as, for example, antigenspecificity, affinity to antigen, potency, biological function, epitoperecognition, stability, solubility, production efficiency,immunogenicity, pharmacokinetics, bioavailability, tissue crossreactivity, or orthologous antigen binding. See, e.g., US PatentPublication No. 20090311253.

The multi-specific antibodies may also be designed such that one or moreof the antigen binding domain are rendered non-functional. The variabledomains may be obtained using recombinant DNA techniques from parentbinding proteins generated by any one of the methods described herein.In an embodiment, a variable domain is a murine heavy or light chainvariable domain. In another embodiment, a variable domain is a CDRgrafted or a humanized variable heavy or light chain domain. In anembodiment, a variable domain is a human heavy or light chain variabledomain.

In an embodiment, one or more constant domains are linked to thevariable domains using recombinant DNA techniques. In an embodiment, asequence comprising one or more heavy chain variable domains is linkedto a heavy chain constant domain and a sequence comprising one or morelight chain variable domains is linked to a light chain constant domain.In an embodiment, the constant domains are human heavy chain constantdomains and human light chain constant domains, respectively. In anembodiment, the heavy chain is further linked to an Fc region. The Fcregion may be a native sequence Fc region or a variant Fc region. Inanother embodiment, the Fc region is a human Fc region. In anotherembodiment, the Fc region includes Fc region from IgG1, IgG2, IgG3,IgG4, IgA, IgM, IgE, or IgD.

Additionally, the binding proteins provided herein can be employed fortissue-specific delivery (target a tissue marker and a disease mediatorfor enhanced local PK thus higher efficacy and/or lower toxicity),including intracellular delivery (targeting an internalizing receptorand an intracellular molecule), delivering to inside brain (targetingtransferrin receptor and a CNS disease mediator for crossing theblood-brain barrier). The binding proteins can also serve as a carrierprotein to deliver an antigen to a specific location via binding to anon-neutralizing epitope of that antigen and also to increase thehalf-life of the antigen. Furthermore, the binding proteins can bedesigned to either be physically linked to medical devices implantedinto patients or target these medical devices (see Burke et al. (2006)Advanced Drug Deliv. Rev. 58(3): 437-446; Hildebrand et al. (2006)Surface and Coatings Technol. 200(22-23): 6318-6324; Drug/devicecombinations for local drug therapies and infection prophylaxis, Wu(2006) Biomaterials 27(11):2450-2467; Mediation of the cytokine networkin the implantation of orthopedic devices, Marques (2005) BiodegradableSystems in Tissue Engineer. Regen. Med. 377-397). Directing appropriatetypes of cell to the site of medical implant may promote healing andrestoring normal tissue function. Alternatively, inhibition of mediators(including but not limited to cytokines), released upon deviceimplantation by a receptor antibody fusion protein coupled to or targetto a device is also provided.

In one aspect, a host cell is transformed with the vector disclosedabove. In one embodiment, the host cell is a prokaryotic cell. In afurther embodiment, the host cell is Escherichia coli. In anotherembodiment, the host cell is an eukaryotic cell. In a furtherembodiment, the eukaryotic cell is selected from the group consisting ofprotist cell, animal cell, plant cell and fungal cell. In oneembodiment, the host cell is a mammalian cell including, but not limitedto, 293, COS, NS0, and CHO and; or a fungal cell such as Saccharomycescerevisiae; or an insect cell such as Sf9.

Another aspect of the invention provides a method of producing a bindingprotein disclosed above comprising culturing any one of the host cellsalso disclosed above in a culture medium under conditions sufficient toproduce the binding protein. Preferably 50%-75% of the binding proteinproduced by this method is a dual specific tetravalent binding protein.More preferably 75%-90% of the binding protein produced by this methodis a dual specific tetravalent binding protein. Most preferably 90%-95%of the binding protein produced is a dual specific tetravalent bindingprotein.

Another embodiment provides a binding protein produced according to themethod disclosed above.

One embodiment provides a composition for the release of a bindingprotein wherein the composition comprises a formulation which in turncomprises a crystallized binding protein, as disclosed above and aningredient; and at least one polymeric carrier. Preferably the polymericcarrier is a polymer selected from one or more of the group consistingof: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid),poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutyrate), poly(caprolactone), poly (dioxanone); poly (ethylene glycol), poly((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof. Preferably theingredient is selected from the group consisting of albumin, sucrose,trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin,methoxypolyethylene glycol and polyethylene glycol. Another embodimentprovides a method for treating a mammal comprising the step ofadministering to the mammal an effective amount of the compositiondisclosed above.

The invention also provides a pharmaceutical composition comprising abinding protein, as disclosed above and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers include, but are notlimited to, phosphate buffer or saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like. Moreparticularly, pharmaceutical compositions suitable for injectable useinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (e.g.,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), and suitable mixtures thereof. In some cases, it will bepreferable to include isotonic agents, for example, sugars,polyalcohols, such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In a further embodiment the pharmaceutical composition comprises atleast one additional therapeutic agent for treating a disorder. In oneembodiment, the additional agent is selected from the group consistingof: therapeutic agents, imaging agents, cytotoxic agent, angiogenesisinhibitors (including but not limited to anti-VEGF antibodies orVEGF-trap); kinase inhibitors (including but not limited to KDR andTIE-2 inhibitors); co-stimulation molecule blockers (including but notlimited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-PD-1, anti-CD20);adhesion molecule blockers (including but not limited to anti-LFA-1 Abs,anti-E/L selectin Abs, small molecule inhibitors); anti-cytokineantibody or functional fragment thereof (including but not limited toanti-IL-18, anti-TNF, anti-IL-6/cytokine receptor antibodies);methotrexate; cyclosporin; rapamycin; FK506; detectable label orreporter; a TNF antagonist; an antirheumatic; a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, and a cytokine antagonist.

In another aspect, the invention provides a method for treating a humansubject suffering from a disorder in which the target, or targets,capable of being bound by the binding protein disclosed above isdetrimental, comprising administering to the human subject a bindingprotein disclosed above such that the activity of the target or targetsin the human subject is inhibited and treatment or preventions of thedisorder is achieved. In one embodiment, the disease or disorder is aninflammatory condition, autoimmune disease, or cancer. In oneembodiment, the disease or disorder is selected from the groupcomprising arthritis, osteoarthritis, juvenile chronic arthritis, septicarthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,ulcerative colitis, inflammatory bowel disease, insulin dependentdiabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis,dermatitis scleroderma, graft versus host disease, organ transplantrejection, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadic,polyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia areata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, Yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren's disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, postinfectious interstitial lung disease, gouty arthritis,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycaemia, type B insulin resistancewith acanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,autoimmune neutropaenia, renal disease NOS, glomerulonephritides,microscopic vasulitis of the kidneys, lyme disease, discoid lupuserythematosus, male infertility idiopathic or NOS, sperm autoimmunity,multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmunethrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroiddisease, hyperthyroidism, goitrous autoimmune hypothyroidism(Hashimoto's disease), atrophic autoimmune hypothyroidism, primarymyxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liverdisease, chronic liver diseases, alcoholic cirrhosis, alcohol-inducedliver injury, choleosatatis, idiosyncratic liver disease, Drug-Inducedhepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders (e.g., depression andschizophrenia), Th2 Type and Th1 Type mediated diseases, acute andchronic pain (different forms of pain), and cancers such as lung,breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic orinfectious processes, acute leukemia, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), acute or chronic bacterialinfection, acute pancreatitis, acute renal failure, adenocarcinomas,aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis,allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis,allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateralsclerosis, anemia, angina pectoris, anterior horn cell degeneration,anti cd3 therapy, antiphospholipid syndrome, anti-receptorhypersensitivity reactions, aordic and peripheral aneuryisms, aorticdissection, arterial hypertension, arteriosclerosis, arteriovenousfistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrialflutter, atrioventricular block, B cell lymphoma, bone graft rejection,bone marrow transplant (BMT) rejection, bundle branch block, Burkitt'slymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiactumors, cardiomyopathy, cardiopulmonary bypass inflammation response,cartilage transplant rejection, cerebellar cortical degenerations,cerebellar disorders, chaotic or multifocal atrial tachycardia,chemotherapy associated disorders, chronic myelocytic leukemia (CML),chronic alcoholism, chronic inflammatory pathologies, chroniclymphocytic leukemia (CLL), chronic obstructive pulmonary disease(COPD), chronic salicylate intoxication, colorectal carcinoma,congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culturenegative sepsis, cystic fibrosis, cytokine therapy associated disorders,Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever,dermatitis, dermatologic conditions, diabetes, diabetes mellitus,diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilatedcongestive cardiomyopathy, disorders of the basal ganglia, Down'sSyndrome in middle age, drug-induced movement disorders induced by drugswhich block CNS dopamine receptors, drug sensitivity, eczema,encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,epstein-barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, fungal sepsis, gas gangrene, gastriculcer, glomerular nephritis, graft rejection of any organ or tissue,gram negative sepsis, gram positive sepsis, granulomas due tointracellular organisms, hairy cell leukemia, Hallerrorden-Spatzdisease, hashimoto's thyroiditis, hay fever, heart transplant rejection,hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolyticthrombocytopenic purpura, hemorrhage, hepatitis (A), His bundlearrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignamt Lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmulti. system disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-Hodgkin's lymphoma, occlusion of theabdominal aorta and its branches, occlusive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue.

In another aspect the invention provides a method of treating a patientsuffering from a disorder comprising the step of administering any oneof the binding proteins disclosed above before, concurrent, or after theadministration of a second agent, as discussed above. In a preferredembodiment the second agent is selected from the group consisting ofbudenoside, epidermal growth factor, corticosteroids, cyclosporin,sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine,metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine,balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptorantagonists, anti-IL-1β monoclonal antibodies, anti-IL-6 monoclonalantibodies, growth factors, elastase inhibitors, pyridinyl-imidazolecompounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7,IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF,and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30,CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin,FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen,corticosteroids, prednisolone, phosphodiesterase inhibitors, adenosineagonists, antithrombotic agents, complement inhibitors, adrenergicagents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1β convertingenzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signallinginhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor,sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10,IL-11, IL-13 and TGFβ.

In one embodiment, the pharmaceutical compositions disclosed above areadministered to the subject by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal,and transdermal.

One aspect of the invention provides at least one anti-idiotype antibodyto at least one binding protein of the present invention. Theanti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one complementarily determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework region, or; any portionthereof, that can be incorporated into a binding protein of the presentinvention.

In another embodiment the binding proteins of the invention are capableof binding one or more targets selected from the group consisting ofABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2;AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3;ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2;BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1;BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1(plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1;CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13(MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC);CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC;exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2);CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3(MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2);CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA);CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR);CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38;CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD47, CD48, CD52;CD69; CD70, CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86;CD137, CD138, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CDH1(E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7;CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A(p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (p16INK4a); CDKN2B;CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2;CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7(claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1;COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF);CSF3 (GCSF); CTLA-4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1(SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11(I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3(GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3(GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1;DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1;EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2(Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A;FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13;FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22;FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8;FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584;FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC);GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1;GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10(C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9;HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA;HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4;IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1; IGF1R; IGF2;IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12;IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15;IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP;IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1;IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2;IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27;IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R;IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA;IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2;ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC BoxBP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6;KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type IIkeratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b);LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7(c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9;MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MYC;MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo;NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1;NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2;NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3;NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; PCSK9; P2RX7;PAP; PART1; PATE; PAWR; PCA3; PCNA; PD-1; PD-L1; alpha4beta7, OX40,GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDF8, CGRP, Lingo-1, Factor IXa,Factor X, ICOS, GARP, BTLA, CD160, ROR1, 2B4, KIR, CD27, OX40, CD40L,A2aR, PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2;PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1;PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2(p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2;SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1;SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2;SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1;STATE; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1;TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissuefactor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF;TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21;TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL);TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14(HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand);TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomeraseIia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6;TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5;VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; andZFPM2.

Given their ability to bind to two or more antigens, the bindingproteins of the present invention can be used to detect the antigens(e.g., in a biological sample, such as serum or plasma), using aconventional immunoassay, such as an enzyme linked immunosorbent assays(ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. TheFIT-Ig is directly or indirectly labeled with a detectable substance tofacilitate detection of the bound or unbound antibody. Suitabledetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, *-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include 3H, 14C, 35S, 90Y, 99Tc, 111In,125I, 131I, 177Lu, 166Ho, or 153Sm.

The binding proteins of the invention, in one embodiment, are capable ofneutralizing the activity of the antigens both in vitro and in vivo.Accordingly, such FIT-Igs can be used to inhibit antigen activity, e.g.,in a cell culture containing the antigens, in human subjects or in othermammalian subjects having the antigens with which a binding protein ofthe invention cross-reacts. In another embodiment, the inventionprovides a method for reducing antigen activity in a subject sufferingfrom a disease or disorder in which the antigen activity is detrimental.A binding protein of the invention can be administered to a humansubject for therapeutic purposes.

As used herein, the term “a disorder in which antigen activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of the antigen in a subject suffering from thedisorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Accordingly, a disorder inwhich antigen activity is detrimental is a disorder in which reductionof antigen activity is expected to alleviate the symptoms and/orprogression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of the antigen in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of antigen in serum, plasma, synovialfluid, etc. of the subject). Non-limiting examples of disorders that canbe treated with the binding proteins of the invention include thosedisorders discussed below and in the section pertaining topharmaceutical compositions of the antibodies of the invention.

The FIT-Igs of the invention may bind one antigen or multiple antigens.Such antigens include, but are not limited to, the targets listed in thefollowing databases, which databases are incorporated herein byreference. These target databases include, but are not limited to, thefollowing listings:

-   -   Therapeutic targets        (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);    -   Cytokines and cytokine receptors        (http://www.cytokinewebfacts.com/,        http://www.copewithcytokines.de/cope.cgi, and    -   http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamotou.acjp/CFC/indexR.html);    -   Chemokines        (http://cytokine.medic.kumamoto-u.acjp/CFC/CK/Chemokine.html);    -   Chemokine receptors and GPCRs        (http://csp.medic.kumamotou.ac.jp/CSP/Receptor.html,        http://www.gpcr.org/7tm/);    -   Olfactory Receptors        (http://senselab.med.yale.edu/senselab/ORDB/default.asp);    -   Receptors (http://www.iuphar-db.org/iuphar-rd/list/index.htm);    -   Cancer targets (http://cged.hgcjp/cgi-bin/input.cgi);    -   Secreted proteins as potential antibody targets        (http://spd.cbi.pku.edu.cn/);    -   Protein kinases (http://spd.cbi.pku.edu.cn/), and    -   Human CD markers        (http://content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf)        and (Zola H, 2005 CD molecules 2005: human cell differentiation        molecules Blood, 106:3123-6).

FIT-Igs are useful as therapeutic agents to simultaneously block twodifferent targets to enhance efficacy/safety and/or increase patientcoverage. Such targets may include soluble targets (IL-13 and TNF) andcell surface receptor targets (VEGFR and EGFR). It can also be used toinduce redirected cytotoxicity between tumor cells and T cells (Her2 andCD3) for cancer therapy, or between autoreactive cell and effector cellsfor autoimmune/transplantation, or between any target cell and effectorcell to eliminate disease-causing cells in any given disease.

In addition, FIT-Ig can be used to trigger receptor clustering andactivation when it is designed to target two different epitopes on thesame receptor. This may have benefit in making agonistic andantagonistic anti-GPCR therapeutics. In this case, FIT-Ig can be used totarget two different epitopes on one cell for clustering/signaling (twocell surface molecules) or signaling (on one molecule). Similarly, aFIT-Ig molecule can be designed to trigger CTLA-4 ligation, and anegative signal by targeting two different epitopes (or 2 copies of thesame epitope) of CTLA-4 extracellular domain, leading to down regulationof the immune response. CTLA-4 is a clinically validated target fortherapeutic treatment of a number of immunological disorders. CTLA-4/B7interactions negatively regulate T cell activation by attenuating cellcycle progression, IL-2 production, and proliferation of T cellsfollowing activation, and CTLA-4 (CD152) engagement can down-regulate Tcell activation and promote the induction of immune tolerance. However,the strategy of attenuating T cell activation by agonistic antibodyengagement of CTLA-4 has been unsuccessful since CTLA-4 activationrequires ligation. The molecular interaction of CTLA-4/B7 is in “skewedzipper” arrays, as demonstrated by crystal structural analysis (Stamper2001 Nature 410:608). However none of the currently available CTLA-4binding reagents have ligation properties, including anti-CTLA-4monoclonal antibodies. There have been several attempts to address thisissue. In one case, a cell member-bound single chain antibody wasgenerated, and significantly inhibited allogeneic rejection in mice(Hwang 2002 JI 169:633). In a separate case, artificial APCsurface-linked single-chain antibody to CTLA-4 was generated anddemonstrated to attenuate T cell responses (Griffin 2000 JI 164:4433).In both cases, CTLA-4 ligation was achieved by closely localizedmember-bound antibodies in artificial systems. While these experimentsprovide proof-of-concept for immune down-regulation by triggering CTLA-4negative signaling, the reagents used in these reports are not suitablefor therapeutic use. To this end, CTLA-4 ligation may be achieved byusing a FIT-Ig molecule, which target two different epitopes (or 2copies of the same epitope) of CTLA-4 extracellular domain. Therationale is that the distance spanning two binding sites of an IgG,approximately 150-170 Å, is too large for active ligation of CTLA-4(30-50 Å between 2 CTLA-4 homodimer). However the distance between thetwo binding sites on FIT-Ig (one arm) is much shorter, also in the rangeof 30-50 Å, allowing proper ligation of CTLA-4.

Similarly, FIT-Ig can target two different members of a cell surfacereceptor complex (e.g. IL-12R alpha and beta). Furthermore, FIT-Ig cantarget CR1 and a soluble protein/pathogen to drive rapid clearance ofthe target soluble protein/pathogen.

Additionally, FIT-Igs of the invention can be employed fortissue-specific delivery (target a tissue marker and a disease mediatorfor enhanced local PK thus higher efficacy and/or lower toxicity),including intracellular delivery (targeting an internalizing receptorand a intracellular molecule), delivering to inside brain (targetingtransferrin receptor and a CNS disease mediator for crossing theblood-brain barrier). FIT-Ig can also serve as a carrier protein todeliver an antigen to a specific location via biding to anon-neutralizing epitope of that antigen and also to increase thehalf-life of the antigen. Furthermore, FIT-Ig can be designed to eitherbe physically linked to medical devices implanted into patients ortarget these medical devices (Burke, Sandra E.; Kuntz, Richard E.;Schwartz, Lewis B. Zotarolimus (ABT-578) eluting stents. Advanced DrugDelivery Reviews (2006), 58(3), 437-446; Surface coatings for biologicalactivation and functionalization of medical devices. Hildebrand, H. F.;Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F. Surfaceand Coatings Technology (2006), 200(22-23), 6318-6324; Drug/devicecombinations for local drug therapies and infection prophylaxis. Wu,Peng; Grainger, David W. Biomaterials (2006), 27(11), 2450-2467;Mediation of the cytokine network in the implantation of orthopedicdevices. Marques, A. P.; Hunt, J. A.; Reis, Rui L. Biodegradable Systemsin Tissue Engineering and Regenerative Medicine (2005), 377-397; Page:52

Mediation of the cytokine network in the implantation of orthopedicdevices. Marques, A. P.; Hunt, J. A.; Reis, Rui L. Biodegradable Systemsin Tissue Engineering and Regenerative Medicine (2005), 377-397.)Briefly, directing appropriate types of cell to the site of medicalimplant may promote healing and restoring normal tissue function.Alternatively, inhibition of mediators (including but not limited tocytokines), released upon device implantation by a FIT-Ig coupled to ortarget to a device is also provided. For example, Stents have been usedfor years in interventional cardiology to clear blocked arteries and toimprove the flow of blood to the heart muscle. However, traditional baremetal stents have been known to cause restenosis (re-narrowing of theartery in a treated area) in some patients and can lead to blood clots.Recently, an anti-CD34 antibody coated stent has been described whichreduced restenosis and prevents blood clots from occurring by capturingendothelial progenitor cells (EPC) circulating throughout the blood.Endothelial cells are cells that line blood vessels, allowing blood toflow smoothly. The EPCs adhere to the hard surface of the stent forminga smooth layer that not only promotes healing but prevents restenosisand blood clots, complications previously associated with the use ofstents (Aoji et al. 2005 J Am Coll Cardiol. 45(10):1574-9). In additionto improving outcomes for patients requiring stents, there are alsoimplications for patients requiring cardiovascular bypass surgery. Forexample, a prosthetic vascular conduit (artificial artery) coated withanti-EPC antibodies would eliminate the need to use arteries frompatients legs or arms for bypass surgery grafts. This would reducesurgery and anesthesia times, which in turn will reduce coronary surgerydeaths. FIT-Ig are designed in such a way that it binds to a cellsurface marker (such as CD34) as well as a protein (or an epitope of anykind, including but not limited to lipids and polysaccharides) that hasbeen coated on the implanted device to facilitate the cell recruitment.Such approaches can also be applied to other medical implants ingeneral. Alternatively, FIT-Igs can be coated on medical devices andupon implantation and releasing all FITs from the device (or any otherneed which may require additional fresh FIT-Ig, including aging anddenaturation of the already loaded FIT-Ig) the device could be reloadedby systemic administration of fresh FIT-Ig to the patient, where theFIT-Ig is designed to binds to a target of interest (a cytokine, a cellsurface marker (such as CD34) etc.) with one set of binding sites and toa target coated on the device (including a protein, an epitope of anykind, including but not limited to lipids, polysaccharides and polymers)with the other. This technology has the advantage of extending theusefulness of coated implants.

FIT-Ig molecules of the invention are also useful as therapeuticmolecules to treat various diseases. Such FIT-Ig molecules may bind oneor more targets involved in a specific disease. Examples of such targetsin various diseases are described below.

Many proteins have been implicated in general autoimmune andinflammatory responses, including C5, CCL1 (I-309), CCL11 (eotaxin),CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18(PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23(MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a),CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10(IP-10), CXCL11 (I-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2,CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13(mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1,IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10,IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF,SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5,IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3,C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD,IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4,TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G,CD3Z, CD69, CD80, CD86, CNR1, CTLA-4, CYSLTR1, FCER1A, FCER2, FCGR3A,GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11,CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11,CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2,BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3,DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1,IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R,IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11,IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15,IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA,LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1,TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144).FIT-Igs capable of binding one or more of the targets listed above arealso contemplated.

Allergic asthma is characterized by the presence of eosinophilia, gobletcell metaplasia, epithelial cell alterations, airway hyperreactivity(AHR), and Th2 and Th1 cytokine expression, as well as elevated serumIgE levels. It is now widely accepted that airway inflammation is thekey factor underlying the pathogenesis of asthma, involving a complexinterplay of inflammatory cells such as T cells, B cells, eosinophils,mast cells and macrophages, and of their secreted mediators includingcytokines and chemokines Corticosteroids are the most importantanti-inflammatory treatment for asthma today, however their mechanism ofaction is non-specific and safety concerns exist, especially in thejuvenile patient population. The development of more specific andtargeted therapies is therefore warranted. There is increasing evidencethat IL-13 in mice mimics many of the features of asthma, including AHR,mucus hypersecretion and airway fibrosis, independently of eosinophilicinflammation (Finotto et al., International Immunology (2005), 17(8),993-1007; Padilla et al., Journal of Immunology (2005), 174(12),8097-8105).

IL-13 has been implicated as having a pivotal role in causingpathological responses associated with asthma. The development ofanti-IL-13 monoclonal antibody therapy to reduce the effects of IL-13 inthe lung is an exciting new approach that offers considerable promise asa novel treatment for asthma. However other mediators of differentialimmunological pathways are also involved in asthma pathogenesis, andblocking these mediators, in addition to IL-13, may offer additionaltherapeutic benefit. Such target pairs include, but are not limited to,IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-α(TNF-α). TNF-α may amplify the inflammatory response in asthma and maybe linked to disease severity (McDonnell, et al., Progress inRespiratory Research (2001), 31(New Drugs for Asthma, Allergy and COPD),247-250.). This suggests that blocking both IL-13 and TNF-α may havebeneficial effects, particularly in severe airway disease. In apreferred embodiment the FIT-Ig of the invention binds the targets IL-13and TNFα and is used for treating asthma.

Animal models such as OVA-induced asthma mouse model, where bothinflammation and AHR can be assessed, are known in the art and may beused to determine the ability of various FIT-Ig molecules to treatasthma. Animal models for studying asthma are disclosed in Coffman, etal., Journal of Experimental Medicine (2005), 201(12), 1875-1879; Lloyd,et al., Advances in Immunology (2001), 77, 263-295; Boyce et al.,Journal of Experimental Medicine (2005), 201(12), 1869-1873; andSnibson, et al., Journal of the British Society for Allergy and ClinicalImmunology (2005), 35(2), 146-52. In addition to routine safetyassessments of these target pairs specific tests for the degree ofimmunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43;Descotes, et al., Developments in biological standardization (1992), 7799-102; Hart et al., Journal of Allergy and Clinical Immunology (2001),108(2), 250-257).

Based on the rationale disclosed above and using the same evaluationmodel for efficacy and safety other pairs of targets that FIT-Igmolecules can bind and be useful to treat asthma may be determined.Preferably such targets include, but are not limited to, IL-13 andIL-1beta, since IL-1beta is also implicated in inflammatory response inasthma; IL-13 and cytokines and chemokines that are involved ininflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5;IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 andTGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13and SPRR2b; and IL-13 and ADAM8. The present invention also contemplatesFIT-Igs capable of binding one or more targets involved in asthmaselected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3(GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors,IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A,IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA,IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1,TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18,CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3,JAK1, JAK3, STATE, TBX21, TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2,LTB4R, TB4R2, LTBR, and Chitinase.

Rheumatoid arthritis (RA), a systemic disease, is characterized by achronic inflammatory reaction in the synovium of joints and isassociated with degeneration of cartilage and erosion of juxta-articularbone. Many pro-inflammatory cytokines including TNF, chemokines, andgrowth factors are expressed in diseased joints. Systemic administrationof anti-TNF antibody or sTNFR fusion protein to mouse models of RA wasshown to be anti-inflammatory and joint protective. Clinicalinvestigations in which the activity of TNF in RA patients was blockedwith intravenously administered infliximab (Harriman G, Harper L K,Schaible T F. 1999 Summary of clinical trials in rheumatoid arthritisusing infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58 Suppl1:161-4.), a chimeric anti-TNF monoclonal antibody (mAB), has providedevidence that TNF regulates IL-6, IL-8, MCP-1, and VEGF production,recruitment of immune and inflammatory cells into joints, angiogenesis,and reduction of blood levels of matrix metalloproteinases-1 and -3. Abetter understanding of the inflammatory pathway in rheumatoid arthritishas led to identification of other therapeutic targets involved inrheumatoid arthritis. Promising treatments such as interleukin-6antagonists (MRA), CTLA4Ig (abatacept, Genovese Mc et al 2005 Abataceptfor rheumatoid arthritis refractory to tumor necrosis factor alphainhibition. N Engl J Med. 353:1114-23.), and anti-B cell therapy(rituximab, Okamoto H, Kamatani N. 2004 Rituximab for rheumatoidarthritis. N Engl J Med. 351:1909) have already been tested inrandomized controlled trials over the past year. Other cytokines havebeen identified and have been shown to be of benefit in animal models,including interleukin-15, interleukin-17, and interleukin-18, andclinical trials of these agents are currently under way. Dual-specificantibody therapy, combining anti-TNF and another mediator, has greatpotential in enhancing clinical efficacy and/or patient coverage. Forexample, blocking both TNF and VEGF can potentially eradicateinflammation and angiogenesis, both of which are involved inpathophysiology of RA. Blocking other pairs of targets involved in RAincluding, but not limited to, TNF and IL-18; TNF and IL-12; TNF andIL-23; TNF and IL-1beta; TNF and MIF; TNF and IL-17; and TNF and IL-15with specific FIT-Ig Igs is also contemplated. In addition to routinesafety assessments of these target pairs, specific tests for the degreeof immunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43;Descotes, et al., Developments in biological standardization (1992), 7799-102; Hart et al., Journal of Allergy and Clinical Immunology (2001),108(2), 250-257). Whether a FIT-Ig Ig molecule will be useful for thetreatment of rheumatoid arthritis can be assessed using pre-clinicalanimal RA models such as the collagen-induced arthritis mouse model.Other useful models are also well known in the art (see Brand D D., CompMed. (2005) 55(2):114-22).

The immunopathogenic hallmark of systemic lupus erythematosus (SLE) isthe polyclonal B cell activation, which leads to hyperglobulinemia,autoantibody production and immune complex formation. The fundamentalabnormality appears to be the failure of T cells to suppress theforbidden B cell clones due to generalized T cell dysregulation. Inaddition, B and T-cell interaction is facilitated by several cytokinessuch as IL-10 as well as co-stimulatory molecules such as CD40 andCD40L, B7 and CD28 and CTLA-4, which initiate the second signal. Theseinteractions together with impaired phagocytic clearance of immunecomplexes and apoptotic material, perpetuate the immune response withresultant tissue injury. The following targets may be involved in SLEand can potentially be used for FIT-Ig approach for therapeuticintervention: B cell targeted therapies: CD-20, CD-22, CD-19, CD28, CD4,CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1,HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81,IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4,HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28,CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24,CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1,ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, and NT5E.; co-stimulatorysignals: CTLA-4 or B7.1/B7.2; inhibition of B cell survival: BlyS, BAFF;Complement inactivation: C5; Cytokine modulation: the key principle isthat the net biologic response in any tissue is the result of a balancebetween local levels of proinflammatory or anti-inflammatory cytokines(see Sfikakis P P et al 2005 Curr Opin Rheumatol 17:550-7). SLE isconsidered to be a Th-2 driven disease with documented elevations inserum IL-4, IL-6, IL-10. FIT-Ig Igs capable of binding one or moretargets selected from the group consisting of IL-4, IL-6, IL-10, IFN-a,and TNF-a are also contemplated. Combination of targets discussed abovewill enhance therapeutic efficacy for SLE which can be tested in anumber of lupus preclinical models (see Peng S L (2004) Methods MolMed.; 102:227-72).

Multiple sclerosis (MS) is a complex human autoimmune-type disease witha predominantly unknown etiology. Immunologic destruction of myelinbasic protein (MBP) throughout the nervous system is the major pathologyof multiple sclerosis. MS is a disease of complex pathologies, whichinvolves infiltration by CD4+ and CD8+ T cells and of response withinthe central nervous system. Expression in the CNS of cytokines, reactivenitrogen species and costimulator molecules have all been described inMS. Of major consideration are immunological mechanisms that contributeto the development of autoimmunity. In particular, antigen expression,cytokine and leukocyte interactions, and regulatory T-cells, which helpbalance/modulate other T-cells such as Th1 and Th2 cells, are importantareas for therapeutic target identification.

IL-12 is a proinflammatory cytokine that is produced by APC and promotesdifferentiation of Th1 effector cells. IL-12 is produced in thedeveloping lesions of patients with MS as well as in EAE-affectedanimals. Previously it was shown that interference in IL-12 pathwayseffectively prevents EAE in rodents, and that in vivo neutralization ofIL-12p40 using a anti-IL-12 mAb has beneficial effects in themyelin-induced EAE model in common marmosets.

TWEAK is a member of the TNF family, constitutively expressed in thecentral nervous system (CNS), with pro-inflammatory, proliferative orapoptotic effects depending upon cell types. Its receptor, Fn14, isexpressed in CNS by endothelial cells, reactive astrocytes and neurons.TWEAK and Fn14 mRNA expression increased in spinal cord duringexperimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibodytreatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE inC57BL/6 mice resulted in a reduction of disease severity and leukocyteinfiltration when mice were treated after the priming phase.

One aspect of the invention pertains to FIT-Ig Ig molecules capable ofbinding one or more, preferably two, targets selected from the groupconsisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF,VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. Apreferred embodiment includes a dual-specific anti-IL-12/TWEAK FIT-Ig Igas a therapeutic agent beneficial for the treatment of MS. Severalanimal models for assessing the usefulness of the FIT-Ig molecules totreat MS are known in the art (see Steinman L, et al., (2005) TrendsImmunol. 26(11):565-71; Lublin F D., et al., (1985) Springer SeminImmunopathol. 8(3):197-208; Genain C P, et al., (1997) J Mol Med.75(3):187-97; Tuohy V K, et al., (1999) J Exp Med. 189(7):1033-42; OwensT, et al., (1995) Neurol Clin. 13(1):51-73; and 't Hart B A, et al.,(2005) J Immunol 175(7):4761-8. In addition to routine safetyassessments of these target pairs specific tests for the degree ofimmunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43;Descotes, et al., Developments in biological standardization (1992), 7799-102; Jones R. 2000 Rovelizumab (ICOS Corp). IDrugs. 3 (4):442-6).

The pathophysiology of sepsis is initiated by the outer membranecomponents of both gram-negative organisms (lipopolysaccharide [LPS],lipid A, endotoxin) and gram-positive organisms (lipoteichoic acid,peptidoglycan). These outer membrane components are able to bind to theCD14 receptor on the surface of monocytes. By virtue of the recentlydescribed toll-like receptors, a signal is then transmitted to the cell,leading to the eventual production of the proinflammatory cytokinestumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-I).Overwhelming inflammatory and immune responses are essential features ofseptic shock and play a central part in the pathogenesis of tissuedamage, multiple organ failure, and death induced by sepsis. Cytokines,especially tumor necrosis factor (TNF) and interleukin (IL)-1, have beenshown to be critical mediators of septic shock. These cytokines have adirect toxic effect on tissues; they also activate phospholipase A2.These and other effects lead to increased concentrations ofplatelet-activating factor, promotion of nitric oxide synthase activity,promotion of tissue infiltration by neutrophils, and promotion ofneutrophil activity.

The treatment of sepsis and septic shock remains a clinical conundrum,and recent prospective trials with biological response modifiers (i.e.anti-TNF, anti-MIF) aimed at the inflammatory response have shown onlymodest clinical benefit. Recently, interest has shifted toward therapiesaimed at reversing the accompanying periods of immune suppression.Studies in experimental animals and critically ill patients havedemonstrated that increased apoptosis of lymphoid organs and someparenchymal tissues contribute to this immune suppression, anergy, andorgan system dysfunction. During sepsis syndromes, lymphocyte apoptosiscan be triggered by the absence of IL-2 or by the release ofglucocorticoids, granzymes, or the so-called ‘death’ cytokines tumornecrosis factor alpha or Fas ligand. Apoptosis proceeds viaauto-activation of cytosolic and/or mitochondrial caspases, which can beinfluenced by the pro- and anti-apoptotic members of the Bcl-2 family.In experimental animals, not only can treatment with inhibitors ofapoptosis prevent lymphoid cell apoptosis; it may also improve outcome.Although clinical trials with anti-apoptotic agents remain distant duein large part to technical difficulties associated with theiradministration and tissue targeting, inhibition of lymphocyte apoptosisrepresents an attractive therapeutic target for the septic patient.Likewise, a dual-specific agent targeting both inflammatory mediator anda apoptotic mediator, may have added benefit. One aspect of theinvention pertains to FIT-Ig Igs capable of binding one or more targetsinvolved in sepsis, preferably two targets, selected from the groupconsisting TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS,Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4,IL10, IL1B, NFKB1, PROC, TNFRSF1A, CSF3, IL10, IL1B, IL6, ADORA2A, CCR3,IL10, IL1B, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine,IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy of such FIT-IgIgs for sepsis can be assessed in preclinical animal models known in theart (see Buras J A, et al., (2005) Nat Rev Drug Discov. 4(10):854-65 andCalandra T, et al., (2000) Nat Med. 6(2):164-70).

Chronic neurodegenerative diseases are usually age-dependent diseasescharacterized by progressive loss of neuronal functions (neuronal celldeath, demyelination), loss of mobility and loss of memory. Emergingknowledge of the mechanisms underlying chronic neurodegenerativediseases (e g Alzheimer's disease) show a complex etiology and a varietyof factors have been recognized to contribute to their development andprogression e.g. age, glycemic status, amyloid production andmultimerization, accumulation of advanced glycation-end products (AGE)which bind to their receptor RAGE (receptor for AGE), increased brainoxidative stress, decreased cerebral blood flow, neuroinflammationincluding release of inflammatory cytokines and chemokines, neuronaldysfunction and microglial activation. Thus these chronicneurodegenerative diseases represent a complex interaction betweenmultiple cell types and mediators. Treatment strategies for suchdiseases are limited and mostly constitute either blocking inflammatoryprocesses with non-specific anti-inflammatory agents (e.g.,corticosteroids, COX inhibitors) or agents to prevent neuron loss and/orsynaptic functions. These treatments fail to stop disease progression.Recent studies suggest that more targeted therapies such as antibodiesto soluble A-β peptide (including the A-b oligomeric forms) can not onlyhelp stop disease progression but may help maintain memory as well.These preliminary observations suggest that specific therapies targetingmore than one disease mediator (e.g. A-β and a pro-inflammatory cytokinesuch as TNF) may provide even better therapeutic efficacy for chronicneurodegenerative diseases than observed with targeting a single diseasemechanism (e.g. soluble A-β alone) (see C. E. Shepherd, et al, NeurobiolAging. 2005 Oct. 24; Nelson R B., Curr Pharm Des. 2005; 11:3335; WilliamL. Klein.; Neurochem Int. 2002; 41:345; Michelle C Janelsins, et al., JNeuroinflammation. 2005; 2:23; Soloman B., Curr Alzheimer Res. 2004;1:149; Igor Klyubin, et al., Nat Med. 2005; 11:556-61; Arancio O, etal., EMBO Journal (2004) 1-10; Bornemann K D, et al., Am J Pathol. 2001;158:63; Deane R, et al., Nat Med. 2003; 9:907-13; and Eliezer Masliah,et al., Neuron. 2005; 46:857).

The FIT-Ig molecules of the invention can bind one or more targetsinvolved in Chronic neurodegenerative diseases such as Alzheimer's. Suchtargets include, but are not limited to, any mediator, soluble or cellsurface, implicated in AD pathogenesis e.g. AGE (S100 A, amphoterin),pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MCP 1),molecules that inhibit nerve regeneration (e.g. Nogo, RGM A), moleculesthat enhance neurite growth (neurotrophins) The efficacy of FIT-Igmolecules can be validated in pre-clinical animal models such as thetransgenic mice that over-express amyloid precursor protein or RAGE anddevelop Alzheimer's disease-like symptoms. In addition, FIT-Ig moleculescan be constructed and tested for efficacy in the animal models and thebest therapeutic FIT-Ig can be selected for testing in human patients.FIT-Ig molecules can also be employed for treatment of otherneurodegenerative diseases such as Parkinson's disease. Alpha-Synucleinis involved in Parkinson's pathology. A FIT-Ig capable of targetingalpha-synuclein and inflammatory mediators such as TNF, IL-1, MCP-1 canprove effective therapy for Parkinson's disease and are contemplated inthe invention.

Despite an increase in knowledge of the pathologic mechanisms, spinalcord injury (SCI) is still a devastating condition and represents amedical indication characterized by a high medical need. Most spinalcord injuries are contusion or compression injuries and the primaryinjury is usually followed by secondary injury mechanisms (inflammatorymediators e.g. cytokines and chemokines) that worsen the initial injuryand result in significant enlargement of the lesion area, sometimes morethan 10-fold. These primary and secondary mechanisms in SCI are verysimilar to those in brain injury caused by other means e.g. stroke. Nosatisfying treatment exists and high dose bolus injection ofmethylprednisolone (MP) is the only used therapy within a narrow timewindow of 8 h post injury. This treatment, however, is only intended toprevent secondary injury without causing any significant functionalrecovery. It is heavily criticized for the lack of unequivocal efficacyand severe adverse effects, like immunosuppression with subsequentinfections and severe histopathological muscle alterations. No otherdrugs, biologics or small molecules, stimulating the endogenousregenerative potential are approved, but promising treatment principlesand drug candidates have shown efficacy in animal models of SCI inrecent years. To a large extent the lack of functional recovery in humanSCI is caused by factors inhibiting neurite growth, at lesion sites, inscar tissue, in myelin as well as on injury-associated cells. Suchfactors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A,the scar-associated CSPG (Chondroitin Sulfate Proteoglycans) andinhibitory factors on reactive astrocytes (some semaphorins andephrins). However, at the lesion site not only growth inhibitorymolecules are found but also neurite growth stimulating factors likeneurotrophins, laminin, L1 and others. This ensemble of neurite growthinhibitory and growth promoting molecules may explain that blockingsingle factors, like NogoA or RGM A, resulted in significant functionalrecovery in rodent SCI models, because a reduction of the inhibitoryinfluences could shift the balance from growth inhibition to growthpromotion. However, recoveries observed with blocking a single neuriteoutgrowth inhibitory molecule were not complete. To achieve faster andmore pronounced recoveries either blocking two neurite outgrowthinhibitory molecules e.g. Nogo and RGM A, or blocking an neuriteoutgrowth inhibitory molecule and enhancing functions of a neuriteoutgrowth enhancing molecule e.g. Nogo and neurotrophins, or blocking aneurite outgrowth inhibitory molecule e.g. Nogo and a pro-inflammatorymolecule e.g. TNF, may be desirable (see McGee A W, et al., TrendsNeurosci. 2003; 26:193; Marco Domeniconi, et al., J Neurol Sci. 2005;233:43; Milan Makwanal, et al., FEBS J. 2005; 272:2628; Barry J.Dickson, Science. 2002; 298:1959; Felicia Yu Hsuan Teng, et al., JNeurosci Res. 2005; 79:273; Tara Karnezis, et al., Nature Neuroscience2004; 7, 736; Gang Xu, et al., J. Neurochem. 2004; 91; 1018).

Other FIT-Igs contemplated are those capable of binding target pairssuch as NgR and RGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A;RGM A and RGM B; CSPGs and RGM A; aggrecan, midkine, neurocan, versican,phosphacan, Te38 and TNF-a; Aβ globulomer-specific antibodies combinedwith antibodies promoting dendrite & axon sprouting. Dendrite pathologyis a very early sign of AD and it is known that NOGO A restrictsdendrite growth. One can combine such type of ab with any of theSCI-candidate (myelin-proteins) Ab. Other FIT-Ig targets may include anycombination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo,Lingo-Troy, Lingo-p75, MAG or Omgp. Additionally, targets may alsoinclude any mediator, soluble or cell surface, implicated in inhibitionof neurite e.g Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, solubleA-b, pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MIP 1a),molecules that inhibit nerve regeneration. The efficacy ofanti-nogo/anti-RGM A or similar FIT-Ig molecules can be validated inpre-clinical animal models of spinal cord injury. In addition, theseFIT-Ig molecules can be constructed and tested for efficacy in theanimal models and the best therapeutic FIT-Ig can be selected fortesting in human patients. In addition, FIT-Ig molecules can beconstructed that target two distinct ligand binding sites on a singlereceptor e.g. Nogo receptor which binds three ligand Nogo, Ompg, and MAGand RAGE that binds A-b and S100 A. Furthermore, neurite outgrowthinhibitors e.g. nogo and nogo receptor, also play a role in preventingnerve regeneration in immunological diseases like multiple sclerosisInhibition of nogo-nogo receptor interaction has been shown to enhancerecovery in animal models of multiple sclerosis. Therefore, FIT-Igmolecules that can block the function of one immune mediator eg acytokine like IL-12 and a neurite outgrowth inhibitor molecule eg nogoor RGM may offer faster and greater efficacy than blocking either animmune or an neurite outgrowth inhibitor molecule alone.

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (von Mehren M, et al 2003 Monoclonal antibodytherapy for cancer. Annu Rev Med.; 54:343-69). Antibodies may exertantitumor effects by inducing apoptosis, redirected cytotoxicity,interfering with ligand-receptor interactions, or preventing theexpression of proteins that are critical to the neoplastic phenotype. Inaddition, antibodies can target components of the tumormicroenvironment, perturbing vital structures such as the formation oftumor-associated vasculature. Antibodies can also target receptors whoseligands are growth factors, such as the epidermal growth factorreceptor. The antibody thus inhibits natural ligands that stimulate cellgrowth from binding to targeted tumor cells. Alternatively, antibodiesmay induce an anti-idiotype network, complement-mediated cytotoxicity,or antibody-dependent cellular cytotoxicity (ADCC). The use ofdual-specific antibody that targets two separate tumor mediators willlikely give additional benefit compared to a mono-specific therapy.FIT-Ig Igs capable of binding the following pairs of targets to treatoncological disease are also contemplated: IGF1 and IGF2; IGF1/2 andErb2B; VEGFR and EGFR; CD20 and CD3, CD138 and CD20, CD38 and CD20, CD38& CD138, CD40 and CD20, CD138 and CD40, CD38 and CD40. Other targetcombinations include one or more members of the EGF/erb-2/erb-3 family.Other targets (one or more) involved in oncological diseases that FIT-IgIgs may bind include, but are not limited to those selected from thegroup consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A,IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11,FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21,FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2,IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2,EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1, IGF1R, IL2, VEGF, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3,NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, ENO2, ENO3,FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMAS, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAILCOL4A3, IL8, LAMAS, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kip1), CDKN2A (p16INK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, CTLA-4, OX40,GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDF8, CGRP, Lingo-1, ICOS, GARP, BTLA,CD160, ROR1, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1(thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A(topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin),CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU (clusterin), ERBB2(Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19),KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3(metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2),S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2(mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and TNFAIP2 (B94).

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods provided herein include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

In an embodiment, the antibodies provided herein or antigen-bindingportions thereof, are used to treat cancer or in the prevention ofmetastases from the tumors described herein either when used alone or incombination with radiotherapy and/or other chemotherapeutic agents.

According to another embodiment of the invention, the human immuneeffector cell is a member of the human lymphoid cell lineage. In thisembodiment, the effector cell may advantageously be a human T cell, ahuman B cell or a human natural killer (NK) cell. Advantageously, suchcells will have either a cytotoxic or an apoptotic effect on the targetcell. Especially advantageously, the human lymphoid cell is a cytotoxicT cell which, when activated, exerts a cytotoxic effect on the targetcell. According to this embodiment, then, the recruited activity of thehuman effector cell is this cell's cytotoxic activity.

According to a preferred embodiment, activation of the cytotoxic T cellmay occur via binding of the CD3 antigen as effector antigen on thesurface of the cytotoxic T cell by a bispecific antibody of thisembodiment of the invention. The human CD3 antigen is present on bothhelper T cells and cytotoxic T cells. Human CD3 denotes an antigen whichis expressed on T cells as part of the multimolecular T cell complex andwhich comprises three different chains: CD3-epsilon, CD3-delta andCD3-gamma.

The activation of the cytotoxic potential of T cells is a complexphenomenon which requires the interplay of multiple proteins. The T cellreceptor (“TCR”) protein is a membrane bound disulfide-linkedheterodimer consisting of two different glycoprotein subunits. The TCRrecognizes and binds foreign peptidic antigen which itself has beenbound by a member of the highly diverse class of majorhistocompatibility complex (“MHC”) proteins and has been presented,bound to the MHC, on the surface of antigen presenting cells (“APCs”).

Although the variable TCR binds foreign antigen as outlined above,signaling to the T cell that this binding has taken place depends on thepresence of other, invariant, signaling proteins associated with theTCR. These signaling proteins in associated form are collectivelyreferred to as the CD3 complex, here collectively referred to as the CD3antigen.

The activation of T cell cytotoxicity, then, normally depends first onthe binding of the TCR with an MHC protein, itself bound to foreignantigen, located on a separate cell. Only when this initial TCR-MHCbinding has taken place can the CD3-dependent signaling cascaderesponsible for T cell clonal expansion and, ultimately, T cellcytotoxicity ensue.

However, binding of the human CD3 antigen by the first or second portionof a bispecific antibody of the invention activates T cells to exert acytotoxic effect on other cells in the absence of independent TCR-MHCbinding. This means that T cells may be cytotoxically activated in aclonally independent fashion, i.e., in a manner which is independent ofthe specific TCR clone carried by the T cell. This allows an activationof the entire T cell compartment rather than only specific T cells of acertain clonal identity.

In light of the foregoing discussion, then, an especially preferredembodiment of the invention provides a bispecific antibody in which theeffector antigen is the human CD3 antigen. The bispecific antibodyaccording to this embodiment of the invention may have a total of eithertwo or three antibody variable domains.

According to further embodiments of the invention, other lymphoidcell-associated effector antigens bound by a bispecific antibody of theinvention may be the human CD16 antigen, the human NKG2D antigen, thehuman NKp46 antigen, the human CD2 antigen, the human CD28 antigen orthe human CD25 antigen.

According to another embodiment of the invention, the human effectorcell is a member of the human myeloid lineage. Advantageously, theeffector cell may be a human monocyte, a human neutrophilic granulocyteor a human dendritic cell. Advantageously, such cells will have either acytotoxic or an apoptotic effect on the target cell. Advantageousantigens within this embodiment which may be bound by a bispecificantibody of the invention may be the human CD64 antigen or the humanCD89 antigen.

According to another embodiment of the invention, the target antigen isan antigen which is uniquely expressed on a target cell or effector cellin a disease condition, but which remains either non-expressed,expressed at a low level or non-accessible in a healthy condition.Examples of such target antigens which might be specifically bound by abispecific antibody of the invention may advantageously be selected fromEpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1(mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, βhCG, Lewis-Y, CD20,CD33, CD30, ganglioside GD3, 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1,Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog(Shh), Wue-1, Plasma Cell Antigen, (membrane-bound) IgE, MelanomaChondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor,STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6;desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25,CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS)Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblastactivation antigen), endosialin, EGFRvIII, LG, SAS and CD63.

According to a specific embodiment, the target antigen specificallybound by a bispecific antibody may be a cancer-related antigen, that isan antigen related to a malignant condition. Such an antigen is eitherexpressed or accessible on a malignant cell, whereas the antigen iseither not present, not significantly present, or is not accessible on anon-malignant cell. As such, a bispecific antibody according to thisembodiment of the invention is a bispecific antibody which recruits theactivity of a human immune effector cell against the malignant targetcell bearing the target antigen, or rendering the target antigenaccessible.

Gene Therapy: In a specific embodiment, nucleic acid sequences encodinga binding protein provided herein or another prophylactic or therapeuticagent provided herein are administered to treat, prevent, manage, orameliorate a disorder or one or more symptoms thereof by way of genetherapy. Gene therapy refers to therapy performed by the administrationto a subject of an expressed or expressible nucleic acid. In thisembodiment, the nucleic acids produce their encoded antibody orprophylactic or therapeutic agent provided herein that mediates aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be used inthe methods provided herein. For general reviews of the methods of genetherapy, see Goldspiel et al. (1993) Clin. Pharmacy 12:488-505; Wu andWu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann Rev. Pharmacol.Toxicol. 32:573-596; Mulligan (1993) Science 260:926-932; Morgan andAnderson (1993) Ann Rev. Biochem. 62:191-217; and May (1993) TIBTECH11(5):155-215. Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley &Sons, NY (1993); andKriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY (1990). Detailed description of various methods of genetherapy are disclosed in US Patent Publication No. US20050042664.

Diagnostics: The disclosure herein also provides diagnostic applicationsincluding, but not limited to, diagnostic assay methods, diagnostic kitscontaining one or more binding proteins, and adaptation of the methodsand kits for use in automated and/or semi-automated systems. Themethods, kits, and adaptations provided may be employed in thedetection, monitoring, and/or treatment of a disease or disorder in anindividual. This is further elucidated below.

A. Method of Assay: The present disclosure also provides a method fordetermining the presence, amount or concentration of an analyte, orfragment thereof, in a test sample using at least one binding protein asdescribed herein. Any suitable assay as is known in the art can be usedin the method. Examples include, but are not limited to, immunoassaysand/or methods employing mass spectrometry. Immunoassays provided by thepresent disclosure may include sandwich immunoassays, radioimmunoassay(RIA), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay(ELISA), competitive-inhibition immunoassays, fluorescence polarizationimmunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT),bioluminescence resonance energy transfer (BRET), and homogenouschemiluminescent assays, among others. A chemiluminescent microparticleimmunoassay, in particular one employing the ARCHITECT® automatedanalyzer (Abbott Laboratories, Abbott Park, Ill.), is an example of animmunoassay. Methods employing mass spectrometry are provided by thepresent disclosure and include, but are not limited to MALDI(matrix-assisted laser desorption/ionization) or by SELDI(surface-enhanced laser desorption/ionization).

Methods for collecting, handling, processing, and analyzing biologicaltest samples using immunoassays and mass spectrometry would bewell-known to one skilled in the art, are provided for in the practiceof the present disclosure (US 2009-0311253 A1).

B. Kit: A kit for assaying a test sample for the presence, amount orconcentration of an analyte, or fragment thereof, in a test sample isalso provided. The kit comprises at least one component for assaying thetest sample for the analyte, or fragment thereof, and instructions forassaying the test sample for the analyte, or fragment thereof. The atleast one component for assaying the test sample for the analyte, orfragment thereof, can include a composition comprising a bindingprotein, as disclosed herein, and/or an anti-analyte binding protein (ora fragment, a variant, or a fragment of a variant thereof), which isoptionally immobilized on a solid phase. Optionally, the kit maycomprise a calibrator or control, which may comprise isolated orpurified analyte. The kit can comprise at least one component forassaying the test sample for an analyte by immunoassay and/or massspectrometry. The kit components, including the analyte, bindingprotein, and/or anti-analyte binding protein, or fragments thereof, maybe optionally labeled using any art-known detectable label. Thematerials and methods for the creation provided for in the practice ofthe present disclosure would be known to one skilled in the art (US2009-0311253 A1).

C. Adaptation of Kit and Method: The kit (or components thereof), aswell as the method of determining the presence, amount or concentrationof an analyte in a test sample by an assay, such as an immunoassay asdescribed herein, can be adapted for use in a variety of automated andsemi-automated systems (including those wherein the solid phasecomprises a microparticle), as described, for example, in U.S. Pat. Nos.5,089,424 and 5,006,309, and as commercially marketed, for example, byAbbott Laboratories (Abbott Park, Ill.) as ARCHITECT®. Other platformsavailable from Abbott Laboratories include, but are not limited to,AxSYM®, IMx® (see, for example, U.S. Pat. No. 5,294,404, PRISM®, EIA(bead), and Quantum™ II, as well as other platforms. Additionally, theassays, kits and kit components can be employed in other formats, forexample, on electrochemical or other hand-held or point-of-care assaysystems. The present disclosure is, for example, applicable to thecommercial Abbott Point of Care (i-STAT®, Abbott Laboratories)electrochemical immunoassay system that performs sandwich immunoassays.Immunosensors and their methods of manufacture and operation insingle-use test devices are described, for example in, U.S. Pat. Nos.5,063,081, 7,419,821, and 7,682,833; and US Publication Nos.20040018577, 20060160164 and US 20090311253. It will be readily apparentto those skilled in the art that other suitable modifications andadaptations of the methods described herein are obvious and may be madeusing suitable equivalents without departing from the scope of theembodiments disclosed herein. Having now described certain embodimentsin detail, the same will be more clearly understood by reference to thefollowing examples, which are included for purposes of illustration onlyand are not intended to be limiting.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of non-critical parameters thatcould be changed or modified to yield essentially similar results.

EXAMPLES Example 1 Construction, Expression, Purification, and Analysisof Anti-IL17/IL-20 Fabs-in-Tandem Immunoglobulin (FIT-Ig)

To demonstrate the FIT-Ig technology, we have generated a group ofanti-IL-17/IL-20 FIT-Ig molecules: FIT1-Ig, FIT2-Ig, and FIT3-Ig, all ofwhich contains 3 different polypeptides, as shown in FIG. 1, whereantigen A is IL-17 and antigen B is IL-20. The DNA construct used togenerate FIT-Ig capable of binding IL-17 and IL-20 is illustrated inFIG. 1B. Briefly, parental mAbs included two high affinity antibodies,anti-IL-17 (clone LY) (U.S. Pat. No. 7,838,638) and anti-hIL-20 (clone15D2) (U.S. Patent Application Publication No. US2014/0194599). Togenerate FIT-Ig construct #1, the VL-CL of LY was directly (FIT1-Ig), orthrough a linker of 3 amino acids (FIT2-Ig) or 7 amino acids (FIT3-Ig)fused to the N-terminus of the 15D2 heavy chain (as shown in Table 1).The construct #2 is VH-CH1 of LY, and the 3^(rd) construct is VL-CL of15D2. The 3 constructs for each FIT-Ig were co-transfected in 293 cells,resulting in the expression and secretion of FIT-Ig protein.

We also generated a group of anti-IL-17/IL-20 FIT-Ig molecules: FIT4-Ig,FIT5-Ig, and FIT6-Ig, each of which contains 2 different polypeptides,as shown in FIG. 2. The DNA constructs used to generate FIT-Ig capableof binding IL-17 and IL-20 are illustrated in FIG. 2B, where antigen Ais IL-17 and antigen B is IL-20. Briefly, parental mAbs included twohigh affinity antibodies, anti-IL-17 (clone LY) and anti-hIL-20 (clone15D2). To generate FIT-Ig construct #1, the VL-CL of LY was directly(FIT4-Ig), or through a linker of 3 amino acids (FIT5-Ig) or 7 aminoacids (FIT6-Ig) fused to the N-terminus of the 15D2 heavy chain (asshown in Table 1). To generate FIT-Ig construct #4, the VH-CH1 of LY wasdirectly (FIT4-Ig), or through a linker of 3 amino acids (FIT5-Ig) or 7amino acids (FIT6-Ig) fused to the N-terminus of the 15D2 light chain.The 2 DNA constructs (construct #1 and #4) for each FIT-Ig wereco-transfected in 293 cells, resulting in the expression and secretionof FIT-Ig protein. The detailed procedures of the PCR cloning aredescribed below.

Example 1.1 Molecular Cloning of Anti-IL-17/IL-20 FIT-Ig Molecules

For construct #1 cloning, LY light chain was amplified by PCR usingforward primers annealing on light chain signal sequence and reverseprimers annealing on C-terminus of the light chain. 15D2 heavy chain wasamplified by PCR using forward primers annealing on N-terminus of 15D2VH and reverse primers annealing on C-terminus of CH. These 2 PCRfragments were gel purified and combined by overlapping PCR using signalpeptide and CH primer pair. The combined PCR product was cloned into a293 expression vector, which already contained the human Fc sequence.

TABLE 1 Anti-IL-17/IL-20 FIT-Ig molecules and DNA constructs. FIT-Igmolecule Construct #1 Linker Construct #2 Construct #3 Construct #4FIT1-Ig VL₁₇-CL-VH₂₀- No linker VH₁₇-CH1 VL₂₀-CL CH1-Fc FIT2-IgVL₁₇-CL-linker- GSG VH₁₇-CH1 VL₂₀-CL VH₂₀-CH1-Fc FIT3-Ig VL₁₇-CL-linker-GGGGSGS VH₁₇-CH1 VL₂₀-CL VH₂₀-CH1-Fc FIT4-Ig VL₁₇-CL-VH₂₀- No linkerVH₁₇-CH1-VL₂₀- CH1-Fc CL FIT5-Ig VL₁₇-CL-linker- GSG VH₁₇-CH1-linker-VH₂₀-CH1-Fc VL₂₀-CL FIT6-Ig VL₁₇-CL-linker- GGGGSGS VH₁₇-CH1-linker-VH₂₀-CH1-Fc VL₂₀-CL

For construct #2 cloning, LY VH-CH1 was amplified by PCR using forwardprimers annealing on heavy chain signal peptide and reverse primerannealing on C-terminal of CH1. The PCR product was gel purified beforecloning into 293 expression vector.

For construct #3, 15D2 light chain was amplified by PCR using forwardprimer annealing on N-terminal of light chain signal peptide and reverseprimer annealing on the end of CL. The PCR product was gel purifiedbefore cloning into 293 expression vector.

For construct #4 cloning, LY VH-CH1 was amplified by PCR using forwardprimer annealing on N-terminus of heavy chain signal peptide and reverseprimer annealing on the end of CH1. 15D2 VL was amplified using primersannealing on the end of 15D2 VL. Both PCR products were gel purified andcombined by overlap PCR. The combined PCR product was gel purified andcloned in 293 expression vector. Table 2 shows sequences of PCR primersused for above molecular cloning.

TABLE 2PCR primers used for molecular construction of anti-IL-17/anti-CD20 FIT-IgsP1: 5′ CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAG 3′ SEQ ID NO. 1 P2: SEQ ID NO. 25′GCTGGACCTGAGAGCCTGAACCGCCACCACCACACTCTCCCCTGT TGAAGC 3′ P3: 5′SEQ ID NO. 3 GGTGGTGGCGGTTCAGGCTCTCAGGTCCAGCTTGTGCAATCTGGCG CCGAGG3′P4: 5′ GTCTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG 3′ SEQ ID NO. 4 P5: 5′TAAGCGTACGGTGGCTGCACCATCTGTCTTC 3′ SEQ ID NO. 5 P6: 5′ SEQ ID NO. 6CGGCGCCAGATTGCACAAGCTGGACCTGGCCTGAACCACACTCTCC CCTGTTGAAGCTC3′ P7: 5′SEQ ID NO. 7 GCTGGACCTGAGAGCCTGAACCGCCACCACCACACTCTCCCCTGTT GAAGC3′P8: 5′ SEQ ID NO. 8 GGTGGTGGCGGTTCAGGCTCTCAGGTCCAGCTTGTGCAATCTGGCGCCGAGG3′ P9: 5′ SEQ ID NO. 9TACCTCGGCGCCAGATTGCACAAGCTGGACCTGACACTCTCCCCTG TTGAAGCTCTTTG3′ P10: 5′SEQ ID NO. CATGACACCTTAACAGAGGCCCCAGGTCGTTTTACCTCGGCGCCAG 10ATTGCACAAG3′ P11: 5′ CAATAAGCTTTACATGACACCTTAACAGAGGCCCCAG3′ SEQ ID NO.11 P12: 5′ TCGAGCGGCCGCTCAACAAGATTTGGGCTCAACTTTCTTG3′ SEQ ID NO. 12 P13:SEQ ID NO. 5′GCTGCTGCTGTGGTTCCCCGGCTCGCGATGCGCTATACAGTTGAC 13 ACAGTC3′P14: 5′ SEQ ID NO. GAAGATGAAGACAGATGGTGCAGCCACCGTACGCTTGATCTCTACC 14TTTGTTC 3′

The final sequences of hIL-17/hIL-20 FIT1-Ig, FIT2-Ig, FIT3-Ig, FIT4-Ig,FIT5-Ig, and FIT6-Ig are listed in Table 3.

TABLE 3 Amino acid sequences of anti-IL-17/IL-20 FIT-Ig moleculesSequence Sequence Protein Protein region Identifier 12345678901234567890Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY FIT1-IgNO.: 15 LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGT POLYPEPTIDE #1DFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK* LY VL SEQ IDDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY NO.: 16LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEI K CL SEQ IDRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLinker None 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWV NO.: 18RQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDY YGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP NO.: 20EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK* Anti-IL-17/IL-20 SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV FIT1-Ig NO.: 21RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADE POLYPEPTIDE #2STSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC LY VH SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV NO.: 22RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ GTLVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCAnti-IL-17/IL-20 SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ FIT1-IgNO.: 23 QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT POLYPEPTIDE #3ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC*15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ NO.: 24QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKCL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY FIT2-IgNO.: 25 LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGT POLYPEPTIDE #1DFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* LY VL SEQ IDDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY NO.: 16LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEI K CL SEQ IDRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLinker SEQ ID GSG NO.: 26 15D2 VH SEQ IDQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWV NO.: 18RQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDY YGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP NO.: 20EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK* Anti-IL-17/IL-20 SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV FIT2-Ig NO.: 21RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADE POLYPEPTIDE #2STSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC LY VH SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV NO.: 22RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ GTLVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCAnti-IL-17/IL-20 SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ FIT2-IgNO.: 23 QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT POLYPEPTIDE #3ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC*15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ NO.: 24QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKCL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY FIT3-IgNO.: 27 LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGT POLYPEPTIDE #1DFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGSQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK* LY VL SEQ IDDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY NO.: 16LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEI K CL SEQ IDRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLinker SEQ ID GGGGSGS NO.: 28 15D2 VH SEQ IDQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWV NO.: 18RQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDY YGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP NO.: 20EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK* Anti-IL-17/IL-20 SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV FIT3-Ig NO.: 21RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADE POLYPEPTIDESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ #2GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC LY VH SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV NO.: 22RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ GTLVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCAnti-IL-17/IL-20 SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ FIT3-IgNO.: 23 QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT POLYPEPTIDEISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVA #3APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC*15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ NO.: 24QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKCL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYFIT4-Ig POLYPEPTIDE NO.: 15 LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGT #1DFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK* LY VL SEQ IDDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY NO.: 16LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEI K CL SEQ IDRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLinker None 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWV NO.: 18RQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDY YGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP NO.: 20EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK* Anti-IL-17/IL-20 SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV FIT4-Ig NO.: 29RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADE POLYPEPTIDE #4STSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC*LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV NO.: 22RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ GTLVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCLinker none 15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ NO.: 24QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKCL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYFIT5-Ig POLYPEPTIDE NO.: 25 LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGT #1DFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* LY VL SEQ IDDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY NO.: 16LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEI K CL SEQ IDRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLinker SEQ ID GSG NO.: 26 15D2 VH SEQ IDQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWV NO.: 18RQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDY YGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP NO.: 20EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK* Anti-IL-17/IL-20 SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV FIT5-Ig NO.: 30RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADE POLYPEPTIDE #4STSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV NO.: 22RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ GTLVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCLinker SEQ ID GSG NO.: 26 15D2 VL SEQ IDAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ NO.: 24QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKCL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYFIT6-Ig POLYPEPTIDE NO.: 27 LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGT #1DFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGSQVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK* LY VL SEQ IDDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTY NO.: 16LHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEI K CL SEQ IDRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLinker GGGGSGS 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVNO.: 18 RQAPGQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDY YGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP NO.: 20EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK* Anti-IL-17/IL-20 SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV FIT6-Ig NO.: 31RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADE POLYPEPTIDE #4STSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGGGSGSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* LY VH SEQ IDQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWV NO.: 22RQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ GTLVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV NO.: 19TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCLinker SEQ ID GGGGSGS NO.: 28 15D2 VL SEQ IDAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ NO.: 24QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKCL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO.: 17KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Example 1.2 Expression, Purification, and Analysis of Anti-IL-17/IL-20FIT-Ig Proteins

All DNA constructs of each FIT-Ig were subcloned into pBOS basedvectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 ofeach FIT-Ig (1, 2, and 3), or construct #1 and #4 of each FIT-Ig (4, 5,and 6) were transiently co-expressed using Polyethyleneimine (PEI) in293E cells. Briefly, DNA in FreeStyle™ 293 Expression Medium was mixedwith the PEI with the final concentration of DNA to PEI ratio of 1:2,incubated for 15 min (no more than 20 min) at room temperature, and thenadded to the 293E cells (1.0-1.2×10⁶/ml, cell viability >95%) at 60 μgDNA/120 ml culture. After 6-24 hours culture in shaker, peptone wasadded to the transfected cells at a final concentration of 5%, withshaking at 125 rpm/min., at 37° C., 8% CO₂. On the 6th-7th day,supernatant was harvested by centrifugation and filtration, and FIT-Igprotein was purified using protein A chromatography (Pierce, Rockford,Ill.) according to the manufacturer's instructions. The proteins wereanalyzed by SDS-PAGE and their concentrations determined by A280 and BCA(Pierce, Rockford, Ill.).

For the expression of FIT1-Ig, FIT2-Ig, and FIT3-Ig, different DNA molarratios of the 3 constructs were used, including construct#1:#2:#3=1:1:1, construct #1:#2:#3=1:1.5:1.5, and construct#1:#2:#3=1:3:3 (Table 4). FIT-Ig proteins were purified by protein Achromatography. The purification yield (7-16 mg/L) was consistent withhIgG quantification of the expression medium for each protein. Thecomposition and purity of the purified FIT-Igs were analyzed by SDS-PAGEin both reduced and non-reduced conditions. In non-reduced conditions,FIT-Ig migrated as a single band of approximately 250 KDa. In reducingconditions, each of the FIT-Ig proteins yielded two bands, one higher MWband is construct #1 of approximately 75 KDa, and one lower MW bandcorresponds to both construct #2 and #3 overlapped at approximately 25KDa. The SDS-PAGE showed that each FIT-Ig is expressed as a singlespecies, and the 3 polypeptide chains are efficiently paired to form anIgG-like molecule. The sizes of the chains as well as the full-lengthprotein of FIT-Ig molecules are consistent with their calculatedmolecular mass based on amino acid sequences.

TABLE 4 Expression and SEC analysis of hIL-17/IL-20 FIT-Ig proteinsFIT-Ig DNA ratio: Expression level % Peak monomeric protein Construct1:2:3 (mg/L) fraction by SEC FIT1-Ig 1:1:1 15.16 92.07 1:1.5:1.5 14.7395.49 1:3:3 9.87 97.92 FIT2-Ig 1:1:1 15.59 90.92 1:1.5:1.5 12.61 94.731:3:3 7.03 97.29 FIT3-Ig 1:1:1 15.59 91.47 1:1.5:1.5 15.16 94.08 1:3:37.75 97.57

To further study the physical properties of FIT-Ig in solution, sizeexclusion chromatography (SEC) was used to analyze each protein. For SECanalysis of the FIT-Ig, purified FIT-Ig, in PBS, was applied on a TSKgelSuperSW3000, 300×4.6 mm column (TOSOH). An HPLC instrument, Model U3000(DIONEX) was used for SEC. All proteins were determined using UVdetection at 280 nm and 214 nm. The elution was isocratic at a flow rateof 0.25 mL/min. All 3 FIT-Ig proteins exhibited a single major peak,demonstrating physical homogeneity as monomeric proteins (Table 4). Theratio of construct #1:#2:#3=1:3:3 showed a better monomeric profile bySEC for all 3 FIT-Ig proteins (Table 4).

Table 4 also shows that the expression levels of all the FIT-Ig proteinsare comparable to that of the regular mAbs, indicating that the FIT-Igcan be expressed efficiently in mammalian cells. For the expression ofFIT4-Ig, FIT5-Ig, and FIT6-Ig, the DNA ration of construct #1:#4=1:1,and the expression level were in the range of 1-10 mg/L, and the % Peakmonomeric fraction as determined by SEC was in the range of 58-76%.Based on this particular mAb combination (LY and 15D2), the3-polypeptide FIT-Ig constructs (FIT1-Ig, FIT2-Ig, and FIT3-Ig) showedbetter expression profile than that of the 2-polypeptide FIT-Igconstructs (FIT4-Ig, FIT5-Ig, and FIT6-Ig), therefore FIT1-Ig, FIT2-Ig,and FIT3-Ig were further analyzed for functional properties

Example 1.3 Determination of Antigen Binding Affinity ofAnti-IL-17/IL-20 FIT-Igs

The kinetics of FIT-Ig binding to rhIL-17 and rhIL-20 was determined bysurface plasmon resonance (Table 5) with a Biacore X100 instrument(Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mMNaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. Briefly, goatanti-human IgG Fcγ fragment specific polyclonal antibody (PierceBiotechnology Inc, Rockford, Ill.) was directly immobilized across a CM5research grade biosensor chip using a standard amine coupling kitaccording to manufacturer's instructions. Purified FIT-Ig samples werediluted in HEPES-buffered saline for capture across goat anti-human IgGFc specific reaction surfaces and injected over reaction matrices at aflow rate of 5 μl/min. The association and dissociation rate constants,kon (M-1s-1) and koff (s-1) were determined under a continuous flow rateof 30 μL/min. Rate constants were derived by making kinetic bindingmeasurements at ten different antigen concentrations ranging from 1.25to 1000 nM. The equilibrium dissociation constant (M) of the reactionbetween FIT-Ig and the target proteins was then calculated from thekinetic rate constants by the following formula: KD=koff/kon. Aliquotsof antigen samples were also simultaneously injected over a blankreference and reaction CM surface to record and subtract any nonspecificbinding background to eliminate the majority of the refractive indexchange and injection noise. Surfaces were regenerated with twosubsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of10 μL/min. The anti-Fc antibody immobilized surfaces were completelyregenerated and retained their full capture capacity over twelve cycles.

TABLE 5 Functional characterizations of anti-IL-17/IL-20 FIT-Igmolecules Binding Kinetics by Biacore Neutralization mAb or k_(on)k_(off) K_(d) Potency IC₅₀ FIT-Ig Antigen (M⁻¹ s⁻¹) (s⁻¹) (M) (pM) LYhIL-17 8.24E+5 1.80E−5 2.18E−11 101 FIT1-Ig hIL-17 1.07E+7 3.88E−53.64E−12 102 FIT2-Ig hIL-17 9.24E+6 1.53E−5 1.65E−12 137 FIT3-Ig hIL-178.71E+6 9.58E−6 1.10E−12 146 15D2 hIL-20 1.70E+6 8.30E−5 5.00E−11 50FIT1-Ig hIL-20 1.40E+6 3.82E−5 2.73E−11 54 FIT2-Ig hIL-20 1.80E+63.50E−5 1.95E−11 50 FIT3-Ig hIL-20 1.40E+6 3.82E−5 2.73E−11 72

The Biacore analysis indicated the overall binding parameters of thethree FIT-Igs to hIL-17 and hIL-20 were similar, with the affinities ofthe FIT-Igs being very close to that of the parental mAb LY and 15D2,and there was no lose of binding affinities for either antigen bindingdomains (Table 5).

In addition, tetravalent dual-specific antigen binding of FIT-Ig wasalso analyzed by Biacore. FIT1-Ig was first captured via a goatanti-human Fc antibody on the Biacore sensor chip, and the first antigenwas injected and a binding signal observed. As the FIT1-Ig was saturatedby the first antigen, the second antigen was then injected and thesecond signal observed. This was done either by first injecting IL-17then IL-20 or by first injecting IL-20 followed by IL-17 for FIT2-Ig(FIG. 3). In either sequence, a dual-binding activity was detected, andboth antigen binding was saturated at 25-30 RU. Similar results wereobtained for FIT2-Ig and FIT3-Ig. Thus each FIT-Ig was able to bind bothantigens simultaneously as a dual-specific tetravalent molecule.

The expression profile and dual-binding properties of FIT-Ig clearlydemonstrated that, within the FIT-Ig molecule, both VL-CL pairedcorrectly with their corresponding VH-CH1 to form 2 functional bindingdomains, and expressed as a single monomeric, tetravalent, andbispecific full length FIT-Ig protein. This is in contrast to themultivalent antibody type of molecules (Miller and Presta, U.S. Pat. No.8,722,859), which displayed tetravalent but mono-specific bindingactivities to one target antigen.

Example 1.4 Determination of Biological Activity of Anti-IL-17/IL-20FIT-Ig

The biological activity of FIT-Ig to neutralize IL-17 function wasmeasured using GROα bioassay. Briefly, Hs27 cells were seeded at 10000cells/50 μL/well into 96 well plates. FIT-Ig or anti-IL-17 controlantibody (25 μL) were added in duplicate wells, with startingconcentration at 2.5 nM followed by 1:2 serial dilutions until 5 pM.IL-17A (25 μL) was then added to each well. The final concentration ofIL-17A was 0.3 nM. Cells were incubated at 37° C. for 17 h before cellculture supernatant were collected. Concentrations of GRO-α in cellculture supernatants were measured by human CACL1/GRO alpha Quantikinekit according to the manufacturer's protocol (R&D systems).

The biological activity of FIT-Ig to neutralize IL-20 function wasmeasured using IL-20R⁺ BAF3 cell proliferation assay. Briefly, 25 μL ofrecombinant human IL-20 at 0.8 nM was added to each well of 96-wellplates (the final concentration of IL-20 is 0.2 nM). Anti-IL20 antibodyor FIT-Ig or other control antibody were diluted to 400 nM (workingconcentration was 100 nM) followed by 5-fold serial dilutions and wereadded to 96-well assay plates (25 μL per well). BaF3 cells stablytransfected with IL-20 receptor were then added to each well atconcentration of 10000 cell/well in volume of 50 μL RPMI 1640 plus 10%FBS, Hygromycin B at the concentration of 800 μg/ml, G418 at theconcentration of 800 μg/ml. After 48-hr incubation, 100 μL CellTiter-GloLuminescent buffer were added to each well. Contents were mixed for 2minutes on an orbital shaker to induce cell lysis and plates wereincubated at room temperature for 10 minutes to stabilize luminescentsignal. Luminescence was recorded by SpectraMax M5.

As shown in Table 5, all FIT-Igs were able to neutralize both hIL-20 andhIL-17, with affinities similar to that of the paternal antibodies.Based on functional analysis using both Biacore and cell-basedneutralization assays, it appears that all 3 FIT-Igs fully retain theactivities of the parental mAbs. There was no significant functionaldifferences among the three FIT-Igs, indicating that the linker wasoptional, and that FIT-Ig construct provided sufficient flexibility andspecial dimension to allow dual binding in the absence of a peptidespacer between the 2 Fab binding regions. This is in contrast to DVD-Igtype of molecules, where a linker between the 2 variable domains on eachof the 2 polypeptide chain is required for retaining activities of thelower (2^(nd)) variable domain.

Example 1.5 Stability Study of Anti-IL-17/IL-20 FIT-Ig

FIT1-Ig protein samples in citrate buffer (pH=6.0) were individuallyincubated at constant 4° C., 25° C. and 40° C. for 1 day, 3 days or 7days; Similarly, FIT1-Ig protein samples were freeze-thawed once, twiceor three times. The fractions of intact full monomeric protein of allsamples was detected by SEC-HPLC, with 10 μg of each protein sampleinjected into Ultimate 3000 HPLC equipping Superdex200 5/150 GL at flowrate 0.3 mL/min for 15 min, and data was recorded and analyzed usingChromeleon software supplied by the manufacturer. Table 6 shows thatFIT1-Ig and FIT3-Ig remained full intact monomeric molecule under thesethermo-challenged conditions.

TABLE 6 Stability analysis of FIT-Ig by measuring % full monomericfractions by SEC Temp. Time (° C.) (day) FIT1-Ig FIT3-Ig 4 0 (Starting)98.74 98.60 1 98.09 97.78 3 97.81 97.45 7 97.63 97.65 25 1 99.00 98.26 399.00 98.01 7 98.86 98.53 40 1 98.95 98.50 3 98.94 98.35 7 98.82 98.371X freeze-thaw 98.89 98.21 2X freeze-thaw 95.37 98.21 3X freeze-thaw95.24 98.35

Example 1.6 Solubility Study of Anti-IL-17/IL-20 FIT-Ig

The solubility of FIT1-Ig was analyzed by measuring sign ofprecipitation in the presence of increasing concentration of PEG6000(PEG6000 was purchased from Shanghai lingfeng chemical reagent co. Ltd).Briefly, solubility of protein in the presence of PEG6000 was obtainedas a function of PEG6000 concentration (0, 5%, 10%, 15%, 20%, 25% and30%). The solubility studies were conducted at a temperature of 25° C.at a solution pH of 6.0. Briefly, protein was precipitated by mixingappropriate quantities of buffered stock solutions of the protein, PEGand the buffer to get the desired concentration of the components. Thefinal volume was made up to 200 μl and the concentration of protein wasset at 1.0 mg/mL. The final solutions were mixed well and equilibratedfor 16 h. After equilibration, the solutions were centrifuged at 13000rpm for 10 min to separate the protein precipitate. Protein solubilitywas measured at 280 nm using Spectra Max Plus384 (Molecular Device) andobtained from the absorbance of the supernatant, and calculating theconcentration based on standard curve of protein concentration (FIG.4A). We also analyzed a commercial antibody Rituxan using the sameexperimental method under 3 different pH conditions (FIG. 4B). Itappears that the protein solubility is dependent on the pH conditions,and that the predicted solubility of FIT-Ig would be in the range ofmonoclonal antibodies.

Example 1.7 Pharmacokinetic Study of Anti-IL-17/IL-20 FIT-Ig

Pharmacokinetic properties of FIT1-Ig were assessed in maleSprague-Dawley (SD) rats. FIT-Ig proteins were administered to male SDrats at a single intravenous dose of 5 mg/kg via a jugular cannula orsubcutaneously under the dorsal skin. Serum samples were collected atdifferent time points over a period of 28 days with sampling at 0, 5,15, and 30 min; 1, 2, 4, 8, and 24 hr; and 2, 4, 7, 10, 14, 21, and 28day serial bleeding via tail vein, and analyzed by human IL-17 captureand/or human IL-20 capture ELISAs. Briefly, ELISA plates were coatedwith goat anti-biotin antibody (5 μg/ml, 4° C., overnight), blocked withSuperblock (Pierce), and incubated with biotinylated human IL-17 (IL-17capture ELISA) or IL-20 (IL-20 capture ELISA) at 50 ng/ml in 10%Superblock TTBS at room temperature for 2 h. Serum samples were seriallydiluted (0.5% serum, 10% Superblock in TTBS) and incubated on the platefor 30 min at room temperature. Detection was carried out withHRP-labeled goat anti human antibody and concentrations were determinedwith the help of standard curves using the four parameter logistic fit.Several animals, especially in the subcutaneous group, showed a suddendrop in FIT-Ig concentrations following day 10, probably due todeveloping an anti-human response. These animals were eliminated fromthe final calculations. Values for the pharmacokinetic parameters weredetermined by non-compartmental model using WinNonlin software(Pharsight Corporation, Mountain View, Calif.).

The rat PK study, FIT1-Ig serum concentrations were very similar whendetermined by the two different ELISA methods, indicating that themolecule was intact, and capable of binding both antigens in vivo. UponIV dosing, FIT1-Ig exhibited a bi-phasic pharmacokinetic profile,consisting of a distribution phase followed by an elimination phase,similar to the PK profile of conventional IgG molecules. Thepharmacokinetic parameters calculated based on the two differentanalytical methods were very similar and are shown in Table 7. Clearanceof FIT-Ig was low (^(˜)12 mL/day/kg), with low volumes of distribution(Vss^(˜)130 mL/kg) resulting in a long half-life (T1/2>10 days).Following subcutaneous administration, FIT-Ig absorbed slowly, withmaximum serum concentrations of approximately 26.9 μg/ml reached at 4days post-dose. The terminal half-life was about 11 days and thesubcutaneous bioavailability was close to 100%. As demonstrated by theseresults, the properties of FIT1-Ig are very similar to a conventionalIgG molecule in vivo, indicating a potential for therapeuticapplications using comparable dosing regimens.

The pharmacokinetics study of FIT-Ig has demonstrated a surprisingbreakthrough in the field of multi-specific Ig-like biologicsdevelopment. The rat pharmacokinetic system is commonly used in thepharmaceutical industry for preclinical evaluation of therapeutic mAbs,and it well predicts the pharmacokinetic profile of mAbs in humans. Thelong half-life and low clearance of FIT-Ig will enable its therapeuticutility for chronic indications with less frequent dosing, similar to atherapeutic mAb. In addition, FIT-Ig, being 100-kDa larger than an IgG,seemed to penetrate efficiently into the tissues based on its IgG-likevolume of distribution parameter from the PK study.

TABLE 7 Pharmacokinetics analysis of FIT1-Ig in SD Rats IV PK parametersCL Vss Beta t_(1/2) AUC MRT Unit mL/day/kg mL/kg Day Day*μg/mL Day IL-17ELISA 12.2 131 10.8 411 10.7 IL-20 ELISA 11.9 128 10.8 421 10.7 SC PKparameters T_(max) C_(max) t_(1/2) AUC_(INF) CL/F F Unit Day ug/mL Dayday*ug/mL mL/day/kg % IL-17 ELISA 4.00 26.9 11.0 406 12.4 103.5 IL-20ELISA 4.00 23.1 10.4 350 14.3 86.4

Example 1.8 Stable CHO Cell Line Development Studies of FIT-Ig

It has been observed that FIT-Ig was efficiently expressed intransiently-transfected 293E cells. In order to further determine themanufacturing feasibility of FIT-Ig, stable transfections were carriedout in both CHO-DG44 and CHO-S cell lines, and subsequent cloneselections as well as productivity analysis were performed. Briefly, CHOcells were transfected by electroporation with 8×10⁶ cells in 400 μltransfection solution plus 20 ug DNA (for CHO DG44 cells) or 25 μg DNA(for CHO-S cells) subcloned in Freedom pCHO vector (Life Technologies).The stable cell line selection was done using routine procedures.Briefly, for CHO-DG44 selection, upon transfection, stable pool wasselected (HT/2P/400G, where P is μg/mL Puromycin, G is μg/mL G418), andprotein production was analyzed by IgG ELISA. Top pools were selectedand proceed to amplification for several rounds with increasingconcentration of MTX (50, 100, 200 and 500 nM), followed by analysis ofprotein production by IgG ELISA. The top pools were then selected forsubcloning. For CHO-S cell selection, the first phase selection wasperformed in medium containing 10P/400G/100M (M is nM MTX), followed byanalysis of protein production. Then the top pools were selected andproceed to 2^(nd) phase selection in either 30P/400G/500M or50P/400G/1000M, followed by protein production measurement by ELISA. Thetop pools were then selected for subcloning. For protein productivityanalysis, fully recovered cell pools (viability >90%) were seeded at5×10⁵ viable cells/mL (CHO DG44) or 3×10⁵ viable cells/mL (CHO-S) using30 mL fresh medium (CD FortiCHO™ medium supplemented with 6 mML-glutamine) in 125-mL shake flasks. The cells were incubated on ashaking platform at 37° C., 80% relative humidity, 8% CO2, and 130 rpm.Sample cultures daily or at regular intervals (e.g., on day 0, 3, 5, 7,10, 12, and 14) to determine the cell density, viability, andproductivity until culture viability drops below 50% or day 14 ofculture is reached. After sampling, feed the cultures with glucose asneeded.

The overall process of FIT1-Ig CHO stable cell line development showedfeatures similar to that of a monoclonal antibody development in CHOcells. For example, during DG44 pool analysis under 2P/400G, the VCDcontinued to increase until day 10-12 up to about 1.3E7, whereas cellviability remained above 80% up to day 13-14, and the productivityreached almost 40 mg/mL on day 14 (FIG. 5A). Upon amplification at5P/400G/50M, productivity reached above 50 mg/mL on day 14 (FIG. 5B).For CHO-S cell selection, the titer reached above 200 mg/mL during thephase 1 selection (FIG. 5C), and above 370 mg/mL at the phase 2selection (FIG. 5D). These levels of productivity are similar to whathave been previously observed for regular human mAb development is ourlaboratory, suggesting that FIT-Ig display mAb-like manufacturingfeasibility for commercial applications.

Example 2 Construction, Expression, and Purification of Anti-CD3/CD20Fabs-in-Tandem Immunoglobulin (FIT-Ig)

To demonstrate if a FIT-Ig can bind to cell surface antigens, we havegenerated an anti-CD3/CD20 FIT-Ig molecule FIT7-Ig and FIT8-Ig, which isthe 3-polypeptide construct, as shown in FIG. 1. The construct used togenerate FIT-Ig capable of binding cell surface CD3 and CD20 isillustrated in FIG. 1B. Briefly, parental mAbs include two high affinityantibodies, anti-CD3 (OKT3) and anti-CD20 (Ofatumumab). To generateFIT7-Ig construct #1, the VL-CL of OKT3 was fused directly (FIT7-Ig) orthrough a linker of 7 amino acids linker (FIT8-Ig) to the N-terminus ofthe Ofatumumab heavy chain (as shown in Table 8). The construct #2 isVH-CH1 of OKT3 and the 3^(rd) construct is VL-CL of Ofatumumab. The 3constructs for FIT-Ig were co-transfected in 293 cells, resulting in theexpression and secretion of FIT-Ig proteins. The detailed procedures ofthe PCR cloning are described below:

Example 2.1 Molecular Cloning of Anti-CD3/CD20 FIT-Ig

The molecular cloning method is similar as that for anti-hIL-17/hIL-20FIT-Ig.

TABLE 8 Anti-CD3/CD20 FIT-Ig molecules and constructs. FIT-Ig moleculeConstruct #1 Linker Construct #2 Construct #3 FIT7-Ig VL_(CD3)-CL- Nolinker VH_(CD3)- VL_(CD20)- VH_(CD20)-CH1-Fc CH1 CL FIT8-Ig VL_(CD3)-CL-GGGGSGS VH_(CD3)- VL_(CD20)- linker- CH1 CL VH_(CD20)-CH1-Fc

Table 9 shows sequences of PCR primers used for molecular constructionabove.

TABLE 9 PCR primers used for molecular construction ofanti-IL-17/IL-20 FIT-Igs SEQ ID NO.P4: GTCTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG 32P12: TCGAGCGGCCGCTCAACAAGATTTGGGCTCAACTTTCTTG 33P20: CAGGTCCAGCTGCAGCAGTCTG 34P22: GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA 35 CAAAGAGCTTCAACAGGGGP23: 36 TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAG P24: 37TGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAP25: CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGAA 38 GTGCAGCTGGTGGAGTCTGP28: 39 GCTGCTGCTGTGGTTCCCCGGCTCGCGATGCGAAATTGTGTTGAC ACAGTC P29: 40AAGATGAAGACAGATGGTGCAGCCACCGTACGTTTAATCTCCAGT CGTGTCC

The final sequences of anti-CD3/CD20 FIT-Ig are described in Table 10.

TABLE 10 Amino acid sequences of anti-CD3/CD20 FIT-Ig Sequence SequenceProtein Protein region Identifier 12345678901234567890 OKT3/OfatumumabSEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY FIT7-Ig NO.: 41QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY POLYPEPTIDE #1SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI NRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLRLSCAASGFTFNDY AMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQY GNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK* OKT3 VL SEQ IDQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY NO.: 42QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR NO.: 17EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Linker none Ofatumumab VH SEQ ID EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHNO.: 43 WVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNY YYGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE NO.: 19PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR NO.: 20TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK* OKT3/OfatumumabSEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH FIT7-Ig NO.: 44WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL POLYPEPTIDE #2TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC LDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC OKT3 VH SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH NO.: 45WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE NO.: 19PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCOKT3/Ofatumumab SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW FIT7-IgNO.: 46 YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD POLYPEPTIDE #3FTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* Ofatumumab VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW NO.: 47YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR NO.: 17EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* OKT3/Ofatumumab SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY FIT8-IgNO.: 48 QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY POLYPEPTIDE #1SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI NRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGSEVQLVESGGGLVQPGRSLRLSCAAS GFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYY CAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK* OKT3 VL SEQ IDQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY NO.: 42QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR NO.: 17EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Linker SEQ ID GGGGSGS NO.: 28 Ofatumumab VHEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMH WVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNY YYGMDVWGQGTTVTVSS CH1 SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE NO.: 19PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC FcSEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR NO.: 20TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK* OKT3/OfatumumabSEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH FIT8-Ig NO.: 44WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL POLYPEPTIDE #2TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC LDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC OKT3 VH SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH NO.: 45WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE NO.: 19PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCOKT3/Ofatumumab SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW FIT8-IgNO.: 46 YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD POLYPEPTIDE #3FTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* Ofatumumab VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW NO.: 47YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR NO.: 17EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Example 2.2 Expression and Purification of Anti-CD3/CD20 FIT-Ig

All DNA constructs of each FIT-Ig were subcloned into pBOS basedvectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 ofeach FIT-Ig were transiently co-expressed using Polyethyleneimine (PEI)in 293E cells. Briefly, DNA in FreeStyle™ 293 Expression Medium wasmixed with the PEI with the final concentration of DNA to PEI ratio of1:2, incubated for 15 min (no more than 20 min) at room temperature, andthen added to the 293E cells (1.0-1.2×10⁶/ml, cell viability >95%) at 60μg DNA/120 ml culture. After 6-24 hours culture in shaker, add peptoneto the transfected cells at a final concentration of 5%, with shaking at125 rpm/min., at 37° C., 8% CO₂. On the 6th-7th day, supernatant washarvested by centrifugation and filtration, and FIT-Ig protein purifiedusing protein A chromatography (Pierce, Rockford, Ill.) according tomanufacturer's instructions. The proteins were analyzed by SDS-PAGE andtheir concentration determined by A280 and BCA (Pierce, Rockford, Ill.)(Table 11).

TABLE 11 Expression and SEC analysis of anti-CD3/CD20 FIT-Ig proteinsFIT-Ig DNA ratio: Expression level % Peak monomeric protein Construct1:2:3 (mg/L) fraction by SEC FIT7-Ig 1:3:3 21.3 99.53 FIT8-Ig 1:3:3 25.699.16

Example 2.3 Binding Activities of Anti-CD3/CD20 FIT-Ig Molecules

Binding of anti-CD3/CD20 FIT-Igs to both targets were analyzed by FACS,using Jurkat cells that express CD3 on the cell surface, as well as Rajicells that express CD20 on the cell surface. Briefly, 5×10⁵ cells werewashed in ice-cold PBS and blocked with 2% FBS on ice for 1 hr. Cellswere incubated with antibody, FIT-Ig (100 nM), or isotype control on icefor 1 hr and washed 3 times with PBS. Secondary antibody (goatanti-human IgG labeled with Alexa Fluor 488, Invitrogen) were added andincubated with cells on ice for 1 hr in dark followed by three timeswash with PBS. Samples were analyzed in FACs caliber. The cell surfacebinding shows that both FIT7-Ig and FIT8-Ig were able to binding to bothcell surface antigens CD3 and CD20 in a concentration dependent manner.Compared to the binding activities of the parental mAbs, FIT-Ig showed areduced binding intensity to CD3 on Jurkat cells, but an enhancedbinding intensity to CD20 on Raji cells. In all binding studies, FIT7-Igand FIT8-Ig showed similar binding activities to both antigens,indicating the linker did not make a significant impact on its bindingability for FIT8-Ig (Table 12).

TABLE 12 Cell surface antigen binding studies of anti-CD3/CD20 FIT-Igproteins FIT-Ig Antigen Binding Intensity by FACS protein (cell line)(MFI) OKT3 CD3 (Jurkat) 399 FIT7-Ig 159 FIT8-Ig 211 Ofatumumab CD20(Raji) 181 FIT7-Ig 291 FIT8-Ig 274

Example 3 Construction, Expression, and Purification of Anti-TNF/IL-17Fabs-in-Tandem Immunoglobulin (FIT-Ig)

Another FIT-Ig that can bind to human IL-17 and human TNFα (FIT9-Ig) wasalso generated using anti-IL-17 mAb clone LY, and anti-TNF mAbGolimumab, in the 3-polypeptide construct, as shown in FIG. 1. Togenerate FIT9-Ig construct #1, the VL-CL of Golimumab was fused directlyto the N-terminus of LY heavy chain (as shown in Table 13). Theconstruct #2 is VH-CH1 of Golimumab and the 3^(rd) construct is VL-CL ofLY. The 3 constructs for FIT9-Ig were co-transfected in 293 cells,resulting in the expression and secretion of FIT9-Ig proteins. The finalsequences of anti-TNF/IL-17 FIT-Ig are described in Table 14.

Example 3.1 Molecular Cloning of Anti-TNF/IL-17 FIT-Ig

The molecular cloning method is similar as that for anti-hIL-17/hIL-20FIT-Ig.

TABLE 13 Anti-TNF/IL-17 FIT-Ig molecule and constructs. FIT-Ig moleculeConstruct #1 Linker Construct #2 Construct #3 FIT9-Ig VL_(TNF)-CL- Nolinker VH_(TNF)- VL_(IL-17)- VH_(IL-17)-CH1-Fc CH1 CL

TABLE 14 Amino acid sequences of anti-TNF/IL-17 FIT-Ig moleculesSequence Sequence Protein Protein region Identifier 12345678901234567890Anti-IL- SEQ ID NO.: 87 EIVLTQSPATLSLSPGER TNF/IL-17 FIT9-ATLSCRASQSVYSYLA Ig WYQQKPGQAPRLLIYD POLYPEPTIDE ASNRATGIPARFSGSGS #1GTDFTLTISSLEPEDFAV YYCQQRSNWPPFTFGP GTKVDIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECQ VQLVQSGAEVKKPGSS VKVSCKASGYSFTDYHI HWVRQAPGQGLEWMGVINPMYGTTDYNQRFK GRVTITADESTSTAYME LSSLRSEDTAVYYCAR YDYFTGTGVYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCL VKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKV DKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWY VDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQ PREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK* GOLIMUMAB VL SEQ ID NO.: 88 EIVLTQSPATLSLSPGER ATLSCRASQSVYSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGS GTDFTLTISSLEPEDFAV YYCQQRSNWPPFTFGPGTKVDIK CL SEQ ID NO.: 17 RTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDST YSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker None LY VH SEQ ID NO.: 22 QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDY HIHWVRQAPGQGLEW MGVINPMYGTTDYNQR FKGRVTITADESTSTAYMELSSLRSEDTAVYYC ARYDYFTGTGVYWGQ GTLVTVSS CH1 SEQ ID NO.: 19ASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYF PEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVE PKSC Fc SEQ ID NO.: 20DKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESN GQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK* Anti-TNF/IL-17 SEQ ID NO.: 89QVQLVESGGGVVQPGR FIT9-Ig SLRLSCAASGFIFSSYA POLYPEPTIDE MHWVRQAPGNGLEWV#2 AFMSYDGSNKKYADSV KGRFTISRDNSKNTLYL QMNSLRAEDTAVYYC ARDRGIAAGGNYYYYGMDVWGQGTTVTVSSA STKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPK SC GOLIMUMAB VHSEQ ID NO.: 90 QVQLVESGGGVVQPGR SLRLSCAASGFIFSSYA MHWVRQAPGNGLEWVAFMSYDGSNKKYADSV KGRFTISRDNSKNTLYL QMNSLRAEDTAVYYC ARDRGIAAGGNYYYYGMDVWGQGTTVTVSS CH1 SEQ ID NO.: 19 ASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSC Anti-IL- SEQ ID NO.: 91 DIVMTQTPLSLSVTPGQ TNF/IL-17 FIT9-PASISCRSSRSLVHSRG Ig NTYLHWYLQKPGQSPQ POLYPEPTIDE LLIYKVSNRFIGVPDRFS #3GSGSGTDFTLKISRVEA EDVGVYYCSQSTHLPF TFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESV TEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C* LY VL SEQ ID NO.: 16 DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRG NTYLHWYLQKPGQSPQ LLIYKVSNRFIGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPF TFGQGTKLEIK CL SEQ ID NO.: 17 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQS GNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC *

Example 3.2 Expression, Purification, and Analysis of Anti-TNF/IL-17FIT-Ig Proteins

All DNA constructs of each FIT-Ig were subcloned into pBOS basedvectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 ofFIT9-Ig were transiently co-expressed using Polyethyleneimine (PEI) in293E cells as described previously and FIT9-Ig proteins were purified byprotein A chromatography. The expression level was 10-23 mg/L. Thepurified protein was subjected to functional analysis using cell-basedassays for IL-17 (production of GROα by Hs27 cells) and TNF (productionof IL-8 by L929 cells). The neutralization potency of FIT9-Ig againsthuman TNF was 11.6 pM (compared to 15.9 pM by Golimumab in the sameexperiment), as against human IL-17 was 122 pM (compared to 51.5 pM byLY in the same experiment). Overall FIT9-Ig maintained the biologicalactivities of the parental mAbs.

Example 4 Construction, Expression, and Purification of Anti-CTLA-4/PD-1Fabs-in-Tandem Immunoglobulin (FIT-Ig)

Another FIT-Ig that can bind to human CTLA-4 and human PD-1 (FIT10-Ig)was generated using anti-CTLA-4 mAb Ipilimumab, and anti-PD-1 mAbNivolumab, in the 3-polypeptide construct, as shown in FIG. 1. Togenerate FIT10-Ig construct #1, the VL-CL of Ipilimumab was fuseddirectly to the N-terminus of Nivolumab heavy chain (as shown in Table15). The construct #2 is VH-CH1 of Ipilimumab and the 3^(rd) constructis VL-CL of Nivolumab. The 3 constructs for FIT10-Ig were co-transfectedin 293 cells, resulting in the expression and secretion of FIT10-Igproteins.

Example 4.1 Molecular Cloning of Anti-CTLA-4/PD-1 FIT-Ig

The molecular cloning method is similar as that for anti-hIL-17/hIL-20FIT-Ig. The final sequences of anti-CTLA-4/PD-1 FIT-Ig are described inTable 16.

TABLE 15 Anti-CTLA-4/PD-1 FIT-Ig molecule and constructs. FIT-Igmolecule Construct #1 Linker Construct #2 Construct #3 FIT10-IgVL_(CTLA-4)-CL- No linker VH_(CTLA-4)- VL_(PD-1)- VH_(PD-1)-CH1-Fc CH1CL

TABLE 16 Amino acid sequences of anti-CTLA-4/PD-1 FIT-Ig moleculesSequence Sequence Protein Protein region Identifier 12345678901234567890Anti-CTLA- SEQ ID NO.: 92 EIVLTQSPGTLSLSPGER 4/PD-1 FIT10-IgATLSCRASQSVGSSYLA POLYPEPTIDE WYQQKPGQAPRLLIYG #1 AFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQYGSSPWTFGQ GTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECQ VQLVESGGGVVQPGRS LRLDCKASGITFSNSGM HWVRQAPGKGLEWVAVIWYDGSKRYYADSVK GRFTISRDNSKNTLFLQ MNSLRAEDTAVYYCAT NDDYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYF PEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGV EVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQ VYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVM HEALHNHYTQKSLSLSP GK*IPILIMUMAB VL SEQ ID NO.: 93 EIVLTQSPGTLSLSPGER ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYG AFSRATGIPDRFSGSGS GTDFTLTISRLEPEDFA VYYCQQYGSSPWTFGQGTKVEIK CL SEQ ID NO.: 17 RTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDST YSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker None NIVOLUMAB VH SEQ ID NO.: 94 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSG MHWVRQAPGKGLEWV AVIWYDGSKRYYADSV KGRFTISRDNSKNTLFLQMNSLRAEDTAVYYC ATNDDYWGQGTLVTV SS CH1 SEQ ID NO.: 19 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSC Fc SEQ ID NO.: 20 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDP EVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDS DGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* Anti-CTLA- SEQ ID NO.: 95 QVQLVESGGGVVQPGR4/PD-1 FIT10-Ig SLRLSCAASGFTFSSYT POLYPEPTIDE MHWVRQAPGKGLEWV #2TFISYDGNNKYYADSV KGRFTISRDNSKNTLYL QMNSLRAEDTAIYYCA RTGWLGPFDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCL VKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKV DKKVEPKSC IPILIMUMAB VHSEQ ID NO.: 96 QVQLVESGGGVVQPGR SLRLSCAASGFTFSSYT MHWVRQAPGKGLEWVTFISYDGNNKYYADSV KGRFTISRDNSKNTLYL QMNSLRAEDTAIYYCA RTGWLGPFDYWGQGTLVTVSS CH1 SEQ ID NO.: 19 ASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSC Anti-CTLA- SEQ ID NO.: 97 EIVLTQSPATLSLSPGER 4/PD-1 FIT10-IgATLSCRASQSVSSYLA POLYPEPTIDE WYQQKPGQAPRLLIYD #3 ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAV YYCQQSSNWPRTFGQG TKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQD SKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC* Nivolumab VL SEQ ID NO.: 98 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYD ASNRATGIPARFSGSGS GTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQG TKVEIK CL SEQ ID NO.: 17 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQS GNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC*

Example 4.2 Expression, Purification, and Functional Analysis ofAnti-CTLA-4/PD-1 FIT-Ig Proteins

All DNA constructs of each FIT-Ig were subcloned into pBOS basedvectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 ofFIT10-Ig were transiently co-expressed using Polyethyleneimine (PEI) in293E cells as described previously and FIT9-Ig proteins were purified byprotein A chromatography to 98% monomeric full protein. The expressionlevels were up to 43 mg/L. The purified protein was subjected to bindinganalysis using ELISA against recombinant CTLA-4Ig and PD-1. Briefly, forbinding to CTLA-4, human CTLA-4Ig (R&D Systems) was immobilized on96-well plates, followed by routine wash and blocking procedures. ThenFIT-10-Ig or Ipilimumab at various concentrations were added to theplate, followed by incubation and multiple wash steps, and detected withanti-human Fab-HRP. For binding to PD-1, human PD-1 (with a his tag)(R&D Systems) was immobilized on 96-well plates, followed by routinewash and blocking procedures. Then FIT-10-Ig or Nivolumab at variousconcentrations were added to the plate, followed by incubation andmultiple wash steps, and detected with anti-human Fc-HRP (FIG. 6). Itappears that FIT10-Ig was able to bind both CTLA-4 (A) and PD-1 (B) withsimilar activities as the parental mAbs Ipilimumab and Nivolumab,respectively.

In addition, multiple-antigen binding study was done using OctetRed todetermine if FIT10-Ig was able to bind recombinant CTLA-4 and PD-1simultaneously. Briefly, FIT10-Ig was immobilize on AR2G sensor atconcentration of 10 μg/ml, followed by binding of CTLA-4Ig and then PD-1(or PD-1 first, then CTLA-4Ig) in assay buffer (PBS pH 7.4, 0.1% BSA,0.02% Tween), with concentration at 80 nM. At the end of the experiment,the surface was regenerated with 10 mM glycine at pH1.5 five times (FIG.7). This experiment shows that FIT10-Ig was able to bind PD-1 when ithad already bound to CTLA-4, and vice versa, indicating that FIT10-Igwas able to bind both CTLA-4Ig and PD-1 simultaneously.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference in its entirety.

1.-57. (canceled)
 58. A binding protein comprising at least twopolypeptide chains, wherein the polypeptide chains pair to form IgG-likemolecules capable of binding two or more antigens, wherein the firstpolypeptide chain comprises VL_(A), CL, VH_(B), and CH1, wherein VL is alight chain variable domain, CL is a light chain constant domain, VH isa heavy chain variable domain, CH1 is the first constant domain of theheavy chain, A is a first antigen, and B is a second antigen.
 59. Thebinding protein of claim 58, wherein the binding protein comprises threepolypeptide chains, wherein the second polypeptide chain comprisesVH_(A) and CH1, and wherein the third polypeptide chain comprises VL_(B)and CL.
 60. The binding protein of claim 59, wherein the firstpolypeptide chain comprises, from amino to carboxyl terminus, i)VL_(A)-CL-VH_(B)-CH1-Fc or ii) VH_(B)-CH1-VL_(A)-CL-Fc; the secondpolypeptide chain comprises, from amino to carboxyl terminus, VH_(A)-CH1and the third polypeptide chain comprises, from amino to carboxylterminus, VL_(B)-CL.
 61. The binding protein of claim 59, wherein thebinding protein comprises a first polypeptide chain comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 15, 25,27, 41, 48, 87, or 92; a second polypeptide chain comprising an aminoacid sequence according to SEQ ID NO: 21, 44, 89, or 95; and a thirdpolypeptide chain comprising a sequence according to SEQ ID NO: 23, 46,91, or
 97. 62. The binding protein of claim 58, wherein the bindingprotein comprises two polypeptide chains, and wherein the secondpolypeptide chain comprises VH_(A), CH1, VL_(B), and CL.
 63. The bindingprotein of claim 62, wherein the first polypeptide chain comprises, fromamino to carboxyl terminus, i) VL_(A)-CL-VH_(B)-CH1-Fc or ii)VH_(B)-CH1-VLA-CL-Fc; and the second polypeptide chain comprises, fromamino to carboxyl terminus, i) VH_(A)-CH1-VL_(B)-CL orVL_(B)-CL-VH_(A)-CH1.
 64. The binding protein of claim 62, wherein thebinding protein comprises a first polypeptide chain comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 15, 25,and 27, and a second polypeptide chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 29, 30, and
 31. 65.The binding protein of claim 58, wherein the binding protein furthercomprises an Fc.
 66. The binding protein of claim 65, wherein the Fc isthe Fc of human IgG1.
 67. The binding protein of claim 65, wherein theFc is a variant Fc.
 68. The binding protein of claim 58, furthercomprising at least one polypeptide linker.
 69. The binding protein ofclaim 68, wherein the at least one polypeptide linker is selected fromthe group consisting of G, GS, SG, GGS, GSG (SEQ ID NO: 26), SGG, GGG,GGGS, SGGG, GGGGS, GGGGSGS (SEQ ID NO: 28), GGGGSGGS, GGGGSGGGGS,GGGGSGGGGSGGGGS, AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, AKTTPKLGG,SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RADAAP, RADAAPTVS,RADAAAAGGPGS, RADAAAA(G₄S)₄, SAKTTP, SAKTTPKLGG, SAKTTPKLEEGEFSEARV,ADAAP, ADAAPTVSIFPP, TVAAP, TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP,AKTTPPSVTPLAP, AKTTAP, AKTTAPSVYPLAP, ASTKGP, ASTKGPSVFPLAP,GENKVEYAPALMALS, GPAKELTPLKEAKVS, and GHEAAAVMQVQYPAS.
 70. The bindingprotein of claim 58, wherein the binding protein is capable of bindingpairs of cytokines selected from the group consisting of IL-1α andIL-1β; IL-12 and IL-18, TNFα and IL-23, TNFα and IL-13; TNF and IL-18;TNF and IL-12; TNF and IL-1beta; TNF and MIF; TNF and IL-6, TNF and IL-6Receptor, TNF and IL-17; IL-17 and IL-20; IL-17 and IL-23; TNF andIL-15; TNF and VEGF; VEGFR and EGFR; IL-13 and IL-9; IL-13 and IL-4;IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS; and TNFα and PGE4, IL-13and PED2, TNF and PEG2, CD138 and CD20; CD138 and CD40; CD19 and CD20;CD19 and CD22; CD20 and CD3; CD3 and CD33; CD16 and CD33, CD3 and CD133;CD38 & CD138; CD38 and CD20; CD20 and CD22; CD38 and CD40; CD40 andCD20; CD-8 and IL-6; CSPGs and RGM A; IGF1 and IGF2; IGF1/2 and Erb2B;IGF-1R and EGFR; EGFR and CD13; IGF-1R and ErbB3; EGFR-2 and IGFR;VEGFR-2 and c-Met; VEGF-A and Angiopoietin-2 (Ang-2); IL-12 and TWEAK;IL-13 and IL-1beta; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGpand RGM A; CTLA-4 and BTNO2; PD-1 and CTLA-4; PD-L1 and CTLA-4; LAG-3and CTLA-4; ICOS and CTLA-4; PD-1 and TIM-3; CD137 and CD20; CD137 andEGFR; CD137 and Her-2; CD137 and PD-1; CD137 and PD-L1; CD137 and CD27;CD137 and GITR; CD137 and HVEM; CD137 and OX40; CD27 and HVEM; CD137 andLAG-3; CD137 and TIM-3; CD137 and VISTA; c-Met and PD-L1; Lag-3 andPD-L1; TIM-3 and PD-L1; EGFR and PD-L1; ICOS and PD-L1; Her2 and PD-L1;PD-L1 and PD-L2; PD-1 and PD-L1; OX40 and PD-L1; VEGF and PD-L1; LAG-3and TIM-3; OX40 and PD-1; BTLA and PD-1; VISTA and PD-1; TIM3 and PD-1;LAG-3 and PD-1; LAG-3 and Her-2; LAG-3 and EGFR; LAG-3 and BTLA; LAG-3and VISTA; OX40 and GITR; OX40 and CD27; OX40 and HVEM; GITR and HYEM;EGFR and DLL-4; VEGF and DLL-4; PDGFR and VEGF; EpCAM and CD3; Her2 andCD3; CD19 and CD3; CD19 and CD20; CEA and EGFR; EpCAM and EGFR; PSMA andPSCA; EphA2 and Her2, EpCAM and CD3; EGFR and Her3; CD16a and CD30; CD30and PSMA; EGFR and CD3; CEA and CD3; TROP-2 and HSG; TROP-2 and CD3;VEGF and EGFR; HGF and VEGF; VEGF and VEGF (same or a differentepitope); VEGF and HGF; EGFR and cMet; PDGF and VEGF; ICOS and PD-1,Lag-3 and PD-1, CD47 and CD20, RGM A and RGM B; Te38 and TNFα; TNFα andBlys; TNFα and CD-22; TNFα and CTLA-4 domain; TNFα and GP130; TNFα andIL-12p40; and TNFα and RANK ligand.
 71. The binding protein of claim 58,wherein the binding protein is capable of binding one or more epitopeson CTLA-4 and/or one or more epitopes on PD-1.
 72. The binding proteinof claim 58, wherein the first polypeptide comprisesVL_(A)-CL-(X1)n-VH_(B)-CH1-(X2)n, wherein VL_(A) is the light chainvariable domain of mAb A, CL is a light chain constant domain, X1represents an amino acid or an oligopeptide linker, VH_(B) is the heavychain variable domain of mAb B, CH1 is the first constant domain of theheavy chain, X2 represents an Fc region, or a different dimerizationdomain, and n is 0 or
 1. 73. A pharmaceutical composition comprising thebinding protein of claim 58 and one or more pharmaceutically acceptablecarrier.
 74. The pharmaceutical composition of claim 73, wherein thecomposition further comprises at least one additional therapeutic agentselected from the group consisting of a cytotoxic agent, an angiogenesisinhibitor, a kinase inhibitor, a co-stimulation molecule blocker; anadhesion molecule blocker, an anti-cytokine antibody or functionalfragment thereof, methotrexate, cyclosporin, rapamycin, FK506, adetectable label or reporter, a TNF antagonist, an antirheumatic, amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteriod, an anabolic steroid, an erythropoietin, an immunization,an immunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog, a cytokine, and a cytokineantagonist.
 75. The binding protein of claim 58, wherein the bindingprotein is conjugated to an agent selected from the group consisting ofan immunoadhesion molecule, an imaging agent, a therapeutic agent, and acytotoxic agent.
 76. The binding protein conjugate of claim 75, whereinthe imaging agent is selected from the group consisting of a radiolabel,an enzyme, a fluorescent label, a luminescent label, a bioluminescentlabel, a magnetic label, and biotin.
 77. A diagnostic kit comprising thebinding protein of claim
 58. 78. A method of treating or preventing aninflammatory disease, autoimmune disease, neurodegenerative disease,cancer, sepsis, metabolic disorder, or spinal cord injury in a subjectin need thereof, the method comprising administering to the subject aneffective amount of the binding protein of claim
 58. 79. The method ofclaim 78, wherein the inflammatory disease, autoimmune disease, orneurodegenerative disease is selected from the group consisting ofasthma, rheumatoid arthritis, systemic lupus erythematosus, multiplesclerosis, Alzheimer's disease, or Parkinson's disease.
 80. The methodof claim 78, wherein the autoimmune disease is selected from the groupconsisting of Crohn's disease, psoriasis (including plaque psoriasis),arthritis (including rheumatoid arthritis, psoriatic arthritis,osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis,ankylosing spondylitis, spondylosing arthropathy, systemic lupuserythematosus, uveitis, sepsis, neurodegenerative diseases, neuronalregeneration, spinal cord injury, primary and metastatic cancers, arespiratory disorder asthma, allergic and nonallergic asthma, asthma dueto infection, asthma due to infection with respiratory syncytial virus(RSV), chronic obstructive pulmonary disease (COPD), a conditioninvolving airway inflammation, eosinophilia, fibrosis and excess mucusproduction, cystic fibrosis, pulmonary fibrosis, an atopic disorder,atopic dermatitis, urticaria, eczema, allergic rhinitis, allergicenterogastritis, an inflammatory and/or autoimmune condition of theskin, an inflammatory and/or autoimmune condition of gastrointestinalorgans, inflammatory bowel diseases (IBD), ulcerative colitis, aninflammatory and/or autoimmune condition of the liver, liver cirrhosis,liver fibrosis, liver fibrosis caused by hepatitis B and/or C virus,scleroderma, tumors or cancers, hepatocellular carcinoma, glioblastoma,lymphoma, Hodgkin's lymphoma, a viral infection, a bacterial infection,a parasitic infection, HTLV-1 infection, suppression of expression ofprotective type 1 immune responses, and suppression of expression of aprotective type 1 immune response during vaccination.
 81. A method oftreating a disease selected from the group consisting of rheumatoidarthritis, psoriasis, psoriatic arthritis, osteoporosis, stroke, liverdisease, and oral cancer in a subject in need thereof, the methodcomprising administering a binding protein according to claim 58 to thesubject, wherein the binding protein is capable of binding IL-17 andIL-20.
 82. A method of treating a B cell cancer in a subject in needthereof, the method comprising administering to the subject a bindingprotein according to claim 58, wherein the binding protein is capable ofbinding CD3 and CD20.
 83. The method of claim 82, wherein the B cellcancer is selected from the group consisting of Hodgkin's lymphoma,non-Hodgkin's lymphoma (NHL), precursor B cell lymphoblasticleukemia/lymphoma, mature B cell neoplasms, B cell chronic lymphocyticleukemia/small lymphocytic lymphoma, B cell prolymphocytic leukemia,lymphoplamacytic lymphoma, mantle cell lymphoma, follicular lymphoma,cutaneous follicle center lymphoma, marginal zone B cell lymphoma, hairycell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma,plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferativedisorder, Waldenstrom's macroglobulinemia, and anaplastic large-celllymphoma.
 84. A method of treating autoimmune or inflammatory disease ina subject in need thereof, the method comprising administering to thesubject a binding protein according to claim 58, wherein the bindingprotein is capable of binding TNF and IL-17.
 85. A method of treatingcancer in a subject in need thereof, the method comprising administeringto the subject a binding protein according to claim 58, wherein thebinding protein is capable of binding CTLA-4 and PD-1.
 86. The method ofclaim 85 wherein the cancer is melanoma, renal cancer, prostate cancer,pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer,esophageal cancer, squamous cell carcinoma of the head and neck, livercancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma,glioma, leukemia, lymphoma, and other neoplastic malignancies.